Therapeutic-wound healing compositions and methods for preparing and using same

ABSTRACT

This invention pertains to therapeutic wound healing compositions for protecting and resuscitating mammalian cells. In one embodiment, the therapeutic wound healing composition comprises (a) pymvate, (b) an antioxidant, and (c) a mixture of saturated and unsaturated fatty acids. In another embodiment, the therapeutic wound healing composition comprises (a) pyruvate, (b) lactate, and (c) a mixture of saturated and unsaturated fatty acids. In yet another embodiment, the therapeutic wound healing composition comprises (a) an antioxidant and (b) a mixture of saturated and unsaturated fatty adds. In still yet another embodiment, the therapeutic wound healing composition comprises (a) lactate, (b) an antioxidant, and (c) a mixture of saturated and unsaturated fatty acids. This invention also pertains to wound healing compositions combined with a medicament which is useful for treating injured mammalian cells to form augmented wound healing compositions such as immunostimulating-wound healing compositions, antiviral-wound healing compositions, antikeratolytic-wound healing compositions, anti-inflammatory-wound healing compositions, antifungal-wound healing compositions, acne treating-wound healing compositions, sunscreen-wound healing compositions, dermatological-wound healing compositions, antihistamine-wound healing compositions, antibacterial-wound healing compositions, and bioadhesive-wound healing compositions. This invention also pertains to wound healing compositions combined with a cytotoxic agent to form cytoprotective-wound healing compositions useful for protecting and reducing injury to mammalian cells and to razor cartridges comprising the wound healing compositions. This invention also pertains to methods for preparing and using the wound healing compositions and the topical and ingestible pharmaceutical products in which the therapeutic compositions may be used.

This is a continuation-in-part of U.S. application Ser. No. 08/187,435filed Jan. 27, 1994; now abandoned, which was a continuation of U.S.application Ser. No. 07/798,392 filed Nov. 26, 1991; now abandoned,which was a continuation-in-part of U.S. application Ser. No. 07/663,500filed Mar. 1, 1991, now abandoned.

DETAILED DESCRIPTION OF THE INVENTION

Applicant has discovered therapeutic wound healing compositions forpreventing and reducing injury to mammalian cells and increasing theresuscitation rate of injured mammalian cells. Cells treated with thetherapeutic wound healing compositions of the present invention showdecreased levels of hydrogen peroxide production, increased resistanceto cytotoxic agents, increased rates of proliferation, and increasedviability. Cellular cultures containing the therapeutic wound healingcompositions showed enhanced differentiation and proliferation overcontrol cultures and rapidly formed attachments or tight junctionsbetween the cells to form an epidermal sheet. Wounded mammals treatedwith the therapeutic wound healing compositions show significantlyimproved wound closing and healing over untreated mammals and mammalstreated with conventional healing compositions. The wound healingcompositions may be used alone or in combination with other medicaments.

In Embodiment One (I), the therapeutic wound healing compositions areused alone. In a first aspect of Embodiment One (I.A), the therapeuticwound healing composition comprises (a) pyruvate selected from the groupconsisting of pyruvic acid, pharmaceutically acceptable salts of pyruvicacid, and mixtures thereof, (b) an antioxidant, and (c) a mixture ofsaturated and unsaturated fatty acids wherein the fatty acids are thosefatty acids required for the repair of cellular membranes andresuscitation of mammalian cells. In a second aspect of Embodiment One(I.B), the therapeutic wound healing composition comprises (a) pyruvateselected from the group consisting of pyruvic acid, pharmaceuticallyacceptable salts of pyrovic acid, and mixtures thereof, (b) lactateselected from the group consisting of lactic acid, pharmaceuticallyacceptable salts of lactic acid, and mixtures thereof, and (c) a mixtureof saturated and unsaturated fatty acids wherein the fatty acids arethose fatty acids required for the repair of cellular membranes andresuscitation of mammalian cells. In a third aspect of Embodiment One(I.C), the therapeutic wound healing composition comprises (a) anantioxidant and (b) a mixture of saturated and unsaturated fatty acidswherein the fatty acids are those fatty acids required for the repair ofcellular membranes and resuscitation of mammalian cells. In a fourthaspect of Embodiment One (I.D), the therapeutic wound healingcomposition comprises (a) lactate selected from the group consisting oflactic acid, pharmaceutically acceptable salts of lactic acid, andmixtures thereof, (b) an antioxidant, and (c) a mixture of saturated andunsaturated fatty acids wherein the fatty acids are those fatty acidsrequired for the repair of cellular membranes and resuscitation ofmammalian cells.

In Embodiment Two (II), the therapeutic wound healing compositions ofEmbodiment One (I.A-D) are combined with a medicament (M) which isuseful for treating injured mammalian cells to form augmented woundhealing compositions (I.A-D+M).

In a first aspect of Embodiment Two (I.A-D+M1), the invention pertainsto a therapeutic immunostimulating-wound healing composition whichcomprises a therapeutically effective amount of an immunestimulatingagent (M1) and a wound healing composition of the present invention(I.A-D). Immunostimulating agents can stimulate the immune system in apatient to kill an infecting organism but do not promote the woundhealing process. Wound healing compositions can increase theresuscitation rate of injured mammalian cells and the proliferation rateof new mammalian cells to replace dead cells but do not kill infectingorganisms. The combination of an immunestimulating agent and a woundhealing composition results in an immunostimulating-wound healingcomposition which acts synergistically to enhance wound repair in boththe upper and lower portions of the skin. The therapeuticimmunostimulating-wound healing compositions are superior in stimulatingboth the extent of re-epithelialization as well as the extent of tissueintegrity and leukocyte infiltration in the dermis.

In a second aspect of Embodiment Two (I.A-D+M2), the invention pertainsto a therapeutic antiviral-wound healing composition which comprises atherapeutically effective mount of an antiviral agent (M2) and a woundhealing composition of the present invention (I.A-D). Antiviral agentscan reduce virus titers in a patient but do not promote the woundhealing process. Wound healing compositions can increase theresuscitation rate of injured mammalian cells and the proliferation rateof new mammalian cells to replace dead cells but do not reduce virustiters. The combination of an antiviral agent and a wound healingcomposition results in a therapeutic antiviral-wound healing compositionwhich reduces the size, duration, and severity of oral and vaginalwounds suffered from viruses such as herpes.

In a third aspect of Embodiment Two (I.A-D+M3), the invention pertainsto a therapeutic antikeratolytic-wound healing composition whichcomprises a therapeutically effective amount of an antikeratolytic agent(M3) and a wound healing composition of the present invention (I.A-D).Antikeratolytic agents can reduce scaling and dryness in a patient butdo not promote the wound healing process. Wound healing compositions canincrease the resuscitation rate of injured mammalian cells and theproliferation rate of new mammalian cells to replace dead cells but donot reduce the proliferation of cells. The combination of anantikeratolytic agent and a wound healing composition results in atherapeutic antikeratolytic-wound healing composition which reduces theduration and severity of psoriasis.

In a fourth aspect of Embodiment Two (I.A-D+M4), the invention pertainsto a therapeutic anti-inflammatory-wound healing composition whichcomprises a therapeutically effective amount of an anti-inflammatoryagent (M4) and a wound healing composition of the present invention(I.A-D). Anti-inflammatory agents can reduce inflammation in a patientbut do not promote the wound healing process. Wound healing compositionscan increase the resuscitation rate of injured mammalian cells and theproliferation rate of new mammalian cells to replace dead cells but donot reduce inflammation. The combination of an anti-inflammatory agentand a wound healing composition results in a therapeuticanti-inflammatory-wound healing composition which reduces the durationand severity of intimation.

In a fifth aspect of Embodiment Two (I.A-D+M5), the invention pertainsto a therapeutic antifungal-wound healing composition which comprises atherapeutically effective amount of a first antifungal agent (M5)selected from the group consisting of lactic acid and sorbic acid and awound healing composition of the present invention (I.A-D). Antifungalagents can treat fungal infections in a patient but do not promote thewound healing process. Wound healing compositions can increase theresuscitation rate of injured mammalian cells and the proliferation rateof new mammalian cells to replace dead cells but do not treat fungalinfections. The combination of an antifungal agent and a wound healingcomposition results in a therapeutic antifungal-wound healingcomposition which reduces the duration and severity of fungalinfections. The therapeutic antifungal-wound healing compositions mayfurther comprise a therapeutically effective amount of a secondantifungal agent and an anti-inflammatory agent.

In a sixth aspect of Embodiment Two (I.A-D+M6), the invention pertainsto a therapeutic acne treating-wound healing composition for the topicaltreatment of acne vulgaris which comprises a therapeutically effectiveamount of tretinoin (M6) and a wound healing composition of the presentinvention (I.A-D). Tretinoin is useful for the topical treatment of acnevulgaris but is known to induce excessive redness, edematous blisteringor crusting, and severe local erythema and peeling at the site ofapplication. Wound healing compositions can increase the resuscitationrate of injured mammalian cells and the proliferation rate of newmammalian cells to replace dead cells. The combination of tretinoin anda wound healing composition results in a therapeutic acne treating-woundhealing composition which reduces the duration and severity of acnevulgaris and the irritation associated with tretinoin. This inventionalso relates to methods for employing the therapeutic acnetreating-wound healing compositions to treat wrinkles.

In a seventh aspect of Embodiment Two (I.A-D+M7), the invention pertainsto a therapeutic sunscreen-wound healing composition useful to minimizeand treat sunburn damage which comprises a therapeutically effectiveamount of a sunscreen agent and an anti-inflammatory agent (sunscreenagent and anti-inflammatory agent collectively referred to as M7) and awound healing composition of the present invention (I.A-D). Sunscreenagents can help prevent sunburn by screening ultra violet light but donot heal injured mammalian cells. Anti-inflammatory agents can reduceinflammation (erythema) in a patient but do not promote the woundhealing process. Wound healing compositions can increase theresuscitation rate of injured mammalian cells and the proliferation rateof new mammalian cells to replace dead cells. Wound healing compositionscan also minimize oxygen radical damage from ultra violet light. Thecombination of a sunscreen agent, an anti-inflammatory agent, and awound healing composition results in a therapeutic sunscreen-woundhealing compositions useful for minimizing and treating sunburn damage.The sunscreen-wound healing compositions may optionally contain atherapeutically effective amount of a topical anesthetic to furtherreduce the severity of sunburn.

In an eighth aspect of Embodiment Two (I.A-D+M8), the invention pertainsto a therapeutic dermatological-wound healing composition useful tominimize and treat diaper dermatitis which comprises a therapeuticallyeffective amount of a buffering agent and an anti-inflammatory agent(buffering agent and anti-inflammatory agent collectively referred to asM8) and a wound healing composition of the present invention (I.A-D).Buffering agents can help prevent diaper dermatitis by neutralizingammonia but do not heal injured mammalian cells. Anti-inflammatoryagents can reduce inflammation (erythema) in a patient but do notpromote the wound healing process. Wound healing compositions canincrease the resuscitation rate of injured mammalian cells and theproliferation rate of new mammalian cells to replace dead cells. Thecombination of a buffering agent, an anti-inflammatory agent, and awound healing composition results in a therapeutic dermatological-woundhealing compositions useful for minimizing and treating diaperdermatitis. The dermatological-wound healing compositions may optionallycontain a therapeutically effective amount of a topical antiseptic tofurther reduce the duration and severity of diaper dermatitis.

In a ninth aspect of Embodiment Two (I.A-D+M9), the invention pertainsto a therapeutic antihistamine-wound healing composition which comprisesa therapeutically effective amount of a topical antihistamine agent (M9)and a wound healing composition of the present invention (I.A-D).Topical antihistamines are useful for the topical treatment of skinirritations but are potentially sensitizing. Wound healing compositionscan increase the resuscitation rate of injured mammalian cells and theproliferation rate of new mammalian cells to replace dead cells. Thecombination of a topical antihistamine and a wound healing compositionresults in a therapeutic antihistamine-wound healing composition whichreduces the duration and severity of itching associated with skinirritations and the sensitizing effect associated with topicalantihistamines.

In a tenth aspect of Embodiment Two (I.A-D+M10), the invention pertainsto a therapeutic antibacterial-wound healing composition which comprisesa therapeutically effective amount of an antibacterial agent (M10) and awound healing composition of the present invention (I.A-D).Antibacterial agents are useful for treating bacterial infections but donot promote the wound healing process. Wound healing compositions canincrease the resuscitation rate of injured mammalian cells and theproliferation rate of new mammalian cells to replace dead cells. Thecombination of an antibacterial agent and a wound healing compositionresults in a therapeutic antibacterial-wound healing composition whichreduces the size, duration, and severity of infected wounds.

In an eleventh aspect of Embodiment Two (I.A-D+M11), the inventionpertains to a therapeutic bioadhesive-wound healing composition whichcomprises a bioadhesive agent (M11) and a therapeutically effectiveamount of a wound healing composition of the present invention (I.A-D).The bioadhesive agent comprises a water-swellable but water-insolublefibrous cross-linked material which adheres to live or freshly killedmucous membranes or skin tissues. The combination of a bioadhesive agentand a wound healing composition results in a therapeuticbioadhesive-wound healing composition which can increase theresuscitation rate of injured mammalian cells and the proliferation rateof new mammalian cells to replace dead cells and thereby reduce theduration and severity of wounds. The therapeutic bioadhesive-woundhealing compositions may further comprise medicaments such asimmunostimulating agents, antiviral agents, antikeratolytic agents,anti-inflammatory agents, antifungal agents, tretinoin, sunscreenagents, dermatological agents, topical antihistamine agents,antibacterial agents, cytotoxic agents, and the like.

In Embodiment Three (III), the therapeutic wound healing compositions ofEmbodiment One (I.A-D) are combined with a cytotoxic agent (X) to formcytoprotective-wound healing compositions (I.A-D+X) useful forpreventing and reducing injury to mammalian cells from a medicamenthaving cytotoxic properties. Cells treated with the cytoprotectivecompositions of the present invention show decreased levels of hydrogenperoxide production, increased resistance to cytotoxic agents, increasedrates of proliferation, and increased viability. The wound healingcompositions may be administered to cells concurrently with a cytotoxicagent or the wound healing compositions may be administered to cellsprior to administration of a cytotoxic anticancer agent to selectivelyprotect non-cancerous cells in the presence of cancerous cells. Becausecancerous cells have a rapid metabolism, cancerous cells will rapidlyconsume the protective wound healing composition and will not beprotected by the wound healing compositions when the chemotherapeuticmedicament is subsequently administered.

In another aspect of Embodiment Three (III), the cytoprotective-woundhealing composition (I.E+X) comprises a cytotoxic agent (X) and a woundhealing composition (I.E) comprising: (a) pyruvate selected from thegroup consisting of pyruvic acid, pharmaceutically acceptable salts ofpyruvic acid, and mixtures thereof; and (b) an antioxidant.

In Embodiment Four (IV), the therapeutic wound healing compositions ofEmbodiment One (I.A-D) are affixed to a razor cartridge (R) to formdisposable razor cartridges comprising wound healing compositions(I.A-D+R). The integral wound healing composition delivery system ispreferably in the form of a solid strip of a water-soluble encapsulatingagent comprising the wound healing composition premixed with a polymericdelivery system. Wound healing compositions can increase theresuscitation rate of injured mammalian cells and the proliferation rateof new mammalian cells to replace dead cells. Applicants have found thatbinding a wound healing composition to a razor cartridge results in atherapeutic razor cartridge which can reduce the duration and severityof shaving cuts and nicks.

In another aspect of Embodiment Four (IV), the disposable razorcartridges (IV.F+R) comprise a wound healing composition comprising anantioxidant (F) affixed to a razor cartridge (R).

The term "injured cell" as used herein means a cell that has anyactivity disrupted for any reason. For example, an injured cell may be acell that has injured membranes or damaged DNA, RNA, and ribosomes, forexample, a cell which has (a) injured membranes so that transportthrough the membranes is diminished resulting in an increase in toxinsand normal cellular wastes inside the cell and a decrease in nutrientsand other components necessary for cellular repair inside the cell, (b)an increase in concentration of oxygen radicals inside the cell becauseof the decreased ability of the cell to produce antioxidants andenzymes, or (c) damaged DNA, RNA, and ribosomes which must be repairedor replaced before normal cellular functions can be resumed. The term"resuscitation" of injured mammalian cells as used herein means thereversal of cytotoxicity, the stabilization of the cellular membrane, anincrease in the proliferation rate of the cell, and/or the normalizationof cellular functions such as the secretion of growth factors, hormones,and the like. The term "cytotoxicity" as used herein means a conditioncaused by a cytotoxic agent that injures the cell. Injured cells do notproliferate because injured cells expend all energy on cellular repair.Aiding cellular repair promotes cellular proliferation.

The term "prodrug", as used herein, refers to compounds which undergobiotransformation prior to exhibiting their pharmacological effects. Thechemical modification of drugs to overcome pharmaceutical problems hasalso been termed "drug latentiation." Drug latentiation is the chemicalmodification of a biologically active compound to form a new compoundwhich upon in vivo enzymatic attack will liberate the parent compound.The chemical alterations of the parent compound are such that the changein physicochemical properties will affect the absorption, distributionand enzymatic metabolism. The definition of drug latentiation has alsobeen extended to include nonenzymatic regeneration of the parentcompound. Regeneration takes place as a consequence of hydrolytic,dissociative, and other reactions not necessarily enzyme mediated. Theterms prodrugs, latentiated drugs, and bioreversible derivatives areused interchangeably. By inference, latentiation implies a time lagelement or time component involved in regenerating the bioactive parentmolecule in vivo. The term prodrug is general in that it includeslatentiated drug derivatives as well as those substances which areconverted after administration to the actual substance which combineswith receptors. The term prodrug is a generic term for agents whichundergo biotransformation prior to exhibiting their pharmacologicalactions. In the case where the administered drug is not the activeagent, but rather is biotransformed to the active agent, the term"prodrug" also includes compounds which may not necessarily undergobiotransformation to the administered drug but may undergobiotransformation to the active agent which exhibits the desiredpharmacological effect.

The term "metabolite", as used herein, refers to any substance producedby metabolism or by a metabolic process. "Metabolism", as used herein,refers to the various chemical reactions involved in the transformationof molecules or chemical compounds occurring in tissue and the cellstherein.

I. Wound Healing Compositions

A. Embodiment One (I.A-D)

The cells which may be treated with the therapeutic wound healingcompositions in the present invention are mammalian cells. Althoughapplicant will describe the present therapeutic wound healingcompositions as useful for treating mammalian epidermal keratinocytesand mammalian monocytes, applicant contemplates that the therapeuticwound healing compositions may be used to protect or resuscitate allmammalian cells. Keratinocytes are representative of normal mammaliancells and are the fastest proliferating cells in the body. Thecorrelation between the reaction of keratinocytes to injury and therapyand that of mammalian cells in general is very high. Monocytes arerepresentative of specialized mammalian cells such as the white bloodcells in the immune system and the organ cells in liver, kidney, heart,and brain. The mammalian cells may be treated in vivo and in vitro.

Epidermal keratinocytes are the specialized epithelial cells of theepidermis which synthesize keratin, a scleroprotein which is theprincipal constituent of epidermis, hair, nails, horny tissue, and theorganic matrix of the enamel of teeth. Mammalian epidermal keratinocytesconstitute about 95% of the epidermal cells and together withmelanocytes form the binary system of the epidermis. In its varioussuccessive stages, epidermal keratinocytes are also known as basalcells, prickle cells, and granular cells.

Monocytes are mononuclear phagocytic leukocytes which undergorespiratory bursting and are involved in reactive oxygen mediated damagewithin the epidermis. Leukocytes are white blood cells or corpuscleswhich may be classified into two main groups: granular leukocytes(granulocytes) which are leukocytes with abundant granules in thecytoplasm and nongranular leukocytes (nongranulocytes) which areleukocytes without specific granules in the cytoplasm and which includethe lymphocytes and monocytes. Phagocyte cells are cells which ingestmicroorganisms or other cells and foreign particles. Monocytes are alsoknown as large mononuclear leukocytes, and hyaline or transitionalleukocytes.

Epidermal keratinocytic cells and monocytic cells have multiple oxygengenerating mechanisms and the degree to which each type of mechanismfunctions differs in each type of cell. In monocytes, for example, therespiratory bursting process is more pronounced than in epidermalkeratinocytes. Hence, the components in the therapeutic wound healingcompositions of the present invention may vary depending upon the typesof cells involved in the condition being treated.

As set out above, in a first aspect of Embodiment One (I.A), thetherapeutic wound healing composition for treating mammalian cells,preferably epidermal keratinocytes, comprises (a) pyruvate selected fromthe group consisting of pyruvic acid, pharmaceutically acceptable saltsof pyrovic acid, and mixtures thereof, (b) an antioxidant, and (c) amixture of saturated and unsaturated fatty acids wherein the fatty acidsare those fatty acids required for the repair of cellular membranes andresuscitation of mammalian cells. In a second aspect of Embodiment One(I.B), the therapeutic wound healing composition for treating mammaliancells, preferably epidermal keratinocytes, comprises (a) pyruvateselected from the group consisting of pyruvic acid, pharmaceuticallyacceptable salts of pyrovic acid, and mixtures thereof, (b) lactateselected from the group consisting of lactic acid, pharmaceuticallyacceptable salts of lactic acid, and mixtures thereof, and (c) a mixtureof saturated and unsaturated fatty acids wherein the fatty acids arethose fatty acids required for the repair of cellular membranes andresuscitation of mammalian cells. In a third aspect of Embodiment One(I.C), the therapeutic wound healing composition for treating mammaliancells, preferably epidermal keratinocytes, comprises (a) an antioxidantand (b) a mixture of saturated and unsaturated fatty acids wherein thefatty acids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells. In a fourth aspect ofEmbodiment One (I.D), the therapeutic wound healing composition fortreating mammalian cells, preferably monocytes, comprises (a) lactateselected from the group consisting of lactic acid, pharmaceuticallyacceptable salts of lactic acid, and mixtures thereof, (b) anantioxidant, and (c) a mixture of saturated and unsaturated fatty acidswherein the fatty acids are those fatty acids required for the repair ofcellular membranes and resuscitation of mammalian cells.

Pyruvic acid (2-oxopropanoic acid, α-ketopropionic acid, CH₃ COCOOH) orpyruvate is a fundamental intermediate in protein and carbohydratemetabolism and in the citric acid cycle. The citric acid cycle(tricarboxylic acid cycle, Kreb's cycle) is the major reaction sequencewhich executes the reduction of oxygen to generate adenosinetriphosphate (ATP) by oxidizing organic compounds in respiring tissuesto provide electrons to the transport system. Acetyl coenzyrne A("active acetyl") is oxidized in this process and is thereafter utilizedin a variety of biological processes and is a precursor in thebiosynthesis of many fatty acids and sterols. The two major sources ofacetyl coenzyme A are derived from the metabolism of glucose and fattyacids. Glycolysis consists of a series of transformations wherein eachglucose molecule is transformed in the cellular cytoplasm into twomolecules of pyruvic acid. Pyruvic acid may then enter the mitochondriawhere it is oxidized by coenzyme A in the presence of enzymes andcofactors to acetyl coenzyme A. Acetyl coenzyme A can then enter thecitric acid cycle.

In muscle, pyrovic acid (derived from glycogen) can be reduced to lacticacid during anerobic metabolism which can occur during exercise. Lacticacid is reoxidized and partially retransformed to glycogen during rest.Pyruvate can also act as an antioxidant to neutralize oxygen radicals inthe cell and can be used in the multifunction oxidase system to reversecytotoxicity.

The pyruvate in the present invention may be selected from the groupconsisting of pyruvic acid, pharmaceutically acceptable salts of pyrovicacid, prodrugs of pyruvic acid, and mixtures thereof. In general, thepharmaceutically acceptable salts of pyruvic acid may be alkali saltsand alkaline earth salts. Preferably, the pyruvate is selected from thegroup consisting of pyrovic acid, lithium pyruvate, sodium pyruvate,potassium pyruvate, magnesium pyruvate, calcium pyruvate, zinc pyruvate,manganese pyruvate, methyl pyruvate, α-ketoglutaric acid, and mixturesthereof. More preferably, the pyruvate is selected from the group ofsalts consisting of sodium pyruvate, potassium pyruvate, magnesiumpyruvate, calcium pyruvate, zinc pyruvate, manganese pyruvate, and thelike, and mixtures thereof. Most preferably, the pyruvate is sodiumpyruvate.

The amount of pyruvate present in the therapeutic wound healingcompositions of the present invention is a therapeutically effectiveamount. A therapeutically effective amount of pyruvate is that amount ofpyruvate necessary for the inventive composition to prevent and reduceinjury to mammalian cells or increase the resuscitation rate of injuredmammalian cells. The exact amount of pyruvate is a matter of preferencesubject to such factors as the type of condition being treated as wellas the other ingredients in the composition. In a preferred embodiment,pyruvate is present in the therapeutic wound healing composition in anamount from about 10% to about 50%, preferably from about 20% to about45%, and more preferably from about 25% to about 40%, by weight of thetherapeutic wound healing composition.

L-Lactic acid ((S)-2-hydroxypropanoic acid, (+) α-hydroxypropionic acid,CH₃ CHOHCOOH) or lactate occurs in small quantities in the blood andmuscle fluid of mammals. Lactic acid concentration increases in muscleand blood after vigorous activity. Lactate is a component in thecellular feedback mechanism and inhibits the natural respiratorybursting process of cells thereby suppressing the production of oxygenradicals.

The lactate in the present invention may be selected from the groupconsisting of lactic acid, pharmaceutically acceptable salts of lacticacid, prodrugs of lactic acid, and mixtures thereof. In general, thepharmaceutically acceptable salts of lactic acid may be alkali salts andalkaline earth salts. Preferably, the lactate is selected from the groupconsisting of lactic acid, lithium lactate, sodium lactate, potassiumlactate, magnesium lactate, calcium lactate, zinc lactate, manganeselactate, and the like, and mixtures thereof. More preferably, thelactate is selected from the group consisting of lactic acid, sodiumlactate, potassium lactate, magnesium lactate, calcium lactate, zinclactate, manganese lactate, and mixtures thereof. Most preferably, thelactate is lactic acid.

The amount of lactate present in the therapeutic wound healingcompositions of the present invention is a therapeutically effectiveamount. A therapeutically effective amount of lactate is that amount oflactate necessary for the inventive composition to prevent and reduceinjury to mammalian cells or increase the resuscitation rate of injuredmammalian cells. For an ingestible composition, a therapeuticallyeffective amount of lactate is that amount necessary to suppress therespiratory bursting process of white blood cells to protect andresuscitate the mammalian cells. In general, a therapeutically effectiveamount of lactate in an ingestible composition is from about 5 to about10 times the amount of lactate normally found in serum. The exact amountof lactate is a matter of preference subject to such factors as the typeof condition being treated as well as the other ingredients in thecomposition. In a preferred embodiment, lactate is present in thetherapeutic wound healing composition in an amount from about 10% toabout 50%, preferably from about 20% to about 45%, and more preferablyfrom about 25% to about 40%, by weight of the therapeutic wound healingcomposition.

Antioxidants are substances which inhibit oxidation or suppressreactions promoted by oxygen or peroxides. Antioxidants, especiallylipid-soluble antioxidants, can be absorbed into the cellular membraneto neutralize oxygen radicals and thereby protect the membrane. Theantioxidants useful in the present invention may be selected from thegroup consisting of all forms of Vitamin A including retinal and3,4-didehydroretinal, all forms of carotene such as Alpha-carotene,β-carotene (beta, β-carotene), gamma-carotene, delta-carotene, all formsof Vitamin C (D-ascorbic acid, L-aseorbic acid), all forms of tocopherolsuch as Vitamin E (Alpha-tocopherol,3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltri-decyl)-2H-1-benzopyran-6-ol),β-tocopherol, gamma-tocopherol, delta-tocopherol, tocoquinone,tocotrienol, and Vitamin E esters which readily undergo hydrolysis toVitamin E such as Vitamin E acetate and Vitamin E succinate, andpharmaceutically acceptable Vitamin E salts such as Vitamin E phosphate,prodrugs of Vitamin A, carotene, Vitamin C, and Vitamin E,pharmaceutically acceptable salts of Vitamin A, carotene, Vitamin C, andVitamin E, and the like, and mixtures thereof. Preferably, theantioxidant is selected from the group of lipid-soluble antioxidantsconsisting of Vitamin A, β-carotene, Vitamin E, Vitamin E acetate, andmixtures thereof. More preferably, the antioxidant is Vitamin E orVitamin E acetate. Most preferably, the antioxidant is Vitamin Eacetate.

The amount of antioxidant present in the therapeutic wound healingcompositions of the present invention is a therapeutically effectiveamount. A therapeutically effective amount of antioxidant is that amountof antioxidant necessary for the inventive composition to prevent andreduce injury to mammalian cells or increase the resuscitation rate ofinjured mammalian cells. The exact amount of antioxidant is a matter ofpreference subject to such factors as the type of condition beingtreated as well as the other ingredients in the composition. In apreferred embodiment, the antioxidant is present in the therapeuticwound healing composition in an amount from about 0.1% to about 40%,preferably from about 0.2% to about 30%, and more preferably from about0.5% to about 20%, by weight of the therapeutic wound healingcomposition.

The mixture of saturated and unsaturated fatty acids in the presentinvention are those fatty acids required for the repair of mammaliancellular membranes and the production of new cells. Fatty acids arecarboxylic acid compounds found in animal and vegetable fat and oil.Fatty acids are classified as lipids and are composed of chains of alkylgroups containing from 4 to 22 carbon atoms and 0-3 double bonds andcharacterized by a terminal carboxyl group, --COOH. Fatty acids may besaturated or unsaturated and may be solid, semisolid, or liquid. Themost common saturated fatty acids are butyric acid (C₄), lauric acid(C₁₂), palmitic acid (C₁₆), and stearic acid (C₁₈). Unsaturated fattyacids are usually derived from vegetables and consist of alkyl chainscontaining from 16 to 22 carbon atoms and 0-3 double bonds with thecharacteristic terminal carboxyl group. The most common unsaturatedfatty acids are oleic acid, linoleic acid, and linolenic acid (all C₁₈acids).

In general, the mixture of saturated and unsaturated fatty acidsrequired for the repair of mammalian cellular membranes in the presentinvention may be derived from animal and vegetable fats and waxes,prodrugs of saturated and unsaturated fatty acids useful in the presentinvention, and mixtures thereof. For example, the fatty acids in thetherapeutic wound healing composition may be in the form of mono-, di-,or trigylcerides, or free fatty acids, or mixtures thereof, which arereadily available for the repair of injured cells. Cells produce thechemical components and the energy required for cellular viability andstore excess energy in the form of fat. Fat is adipose tissue storedbetween organs of the body to furnish a reserve supply of energy. Thepreferred animal fats and waxes have a fatty acid composition similar tothat of human fat and the fat contained in human breast milk. Thepreferred animal fats and waxes may be selected from the groupconsisting of human fat, chicken fat, cow fat (defined herein as abovine domestic animal regardless of sex or age), sheep fat, horse fat,pig fat, and whale fat. The more preferred animal fats and waxes may beselected from the group consisting of human fat and chicken fat. Themost preferred animal fat is human fat. Mixtures of other fats andwaxes, such as vegetable waxes (especially sunflower oil), marine oils(especially shark liver oil), and synthetic waxes and oils, which have afatty acid composition similar to that of animal fats and waxes, andpreferably to that of human fats and waxes, may also be employed.

In a preferred embodiment, the mixture of saturated and unsaturatedfatty acids has a composition similar to that of human fat and comprisesthe following fatty acids: butyric acid, caproic acid, caprylic acid,captic acid, laurie acid, myristic acid, myristoleic acid, palmiticacid, palmitoleic acid, stearic, oleic acid, linoleic acid, linolenicacid, arachidic acid, and gadoleic acid. Preferably, butyric acid,caproic acid, caprylic acid, capric acid, laurie acid, myristic acid,myristoleic acid, palmitic acid, palmitoleic acid, stearic, oleic acid,linoleic acid, linolenic acid, arachidic acid, and gadoleic acid arepresent in the mixture in about the following percentages by weight,respectively (carbon chain number and number of unsaturations are shownparenthetically, respectively): 0.2%-0.4% (C₄), 0.1% (C₆), 0.3%-0.8%(C₈), 2.2%-3.5% (C₁₀), 0.9%-5.5% (C₁₂), 2.8%-8.5% (C₁₄), 0.1%-0.6%(C_(14:1)), 23.2%-24.6% (C₁₆), 1.8-%3.0% (C_(16:1)), 6.9%-9.9% (C₁₈),36.0%-36.5% (C_(18:1)), 20%-20.6% (C_(18:2)), 7.5-7.8% (C_(18:3)),1.1%-4.9% (C₂₀), and 3.3%-6.4% (C_(20:1)).

In another preferred embodiment, the mixture of saturated andunsaturated fatty acids is typically chicken fat comprising thefollowing fatty acids: lauric acid, myristic acid, myristoleic acid,pentadecanoic acid, palmitic acid, palmitoleic acid, margaric acid,margaroleic acid, stearic, oleic acid, linoleic acid, linolenic acid,arachidic acid, and gadoleic acid. Preferably, lauric acid, myristicacid, myristoleic acid, pentadecanoic acid, palmitic acid, palmitoleicacid, margaric acid, margaroleic acid, stearic, oleic acid, linoleicacid, linolenic acid, arachidic acid, and gadoleic acid are present inthe mixture in about the following percentages by weight, respectively:0.1% (C₁₂), 0.8% (C₁₄), 0.2% (C_(14:1)), 0.1% (C₁₅), 25.3% (C₁₆), 7.2%(C_(16:1)), 0.1% (C₁₇), 0.1% (C_(17:1)), 6.5% (C₁₈), 37.7% (C_(18:1)),20.6% (C_(18:2)), 0.8% (C_(18:3)), 0.2% (C₂₀), and 0.3% (C_(20:1)), allpercentages ±10%.

In another preferred embodiment, the mixture of saturated andunsaturated fatty acids comprises lecithin. Lecithin(phosphatidylcholine) is a phosphatide found in all living organisms(plants and animals) and is a significant constituent of nervous tissueand brain substance. Lecithin is a mixture of the diglycerides ofstearic, palmitic, and oleic acids, linked to the oholine ester ofphosphoric acid. The product of commerce is predominantly soybeanlecithin obtained as a by-product in the manufacturing of soybean oil.Soybean lecithin contains palmitic acid 11.7%, stearic 4.0%, palmitoleic8.6%, oleic 9.8%, linoleic 55.0%, linolenic 4.0%, C₂₀ to C₂₂ acids(includes arachidonic) 5.5%. Lecithin may be represented by the formula:##STR1## wherein R is selected from the group consisting of stearic,palmitic, and oleic acid.

The above fatty acids and percentages thereof present in the fatty acidmixture are given as an example. The exact type of fatty acid present inthe fatty acid mixture and the exact amount of fatty acid employed inthe fatty acid mixture may be varied in order to obtain the resultdesired in the final product and such variations are now within thecapabilities of those skilled in the art without the need for undueexperimentation.

The amount of fatty acids present in the therapeutic wound healingcompositions of the present invention is a therapeutically effectiveamount. A therapeutically effective amount of fatty acids is that amountof fatty acids necessary for the inventive composition to prevent andreduce injury to mammalian cells or increase the resuscitation rate ofinjured mammalian cells. The exact amount of fatty acids employed issubject to such factors as the type and distribution of fatty acidsemployed in the mixture, the type of condition being treated, and theother ingredients in the composition. In a preferred embodiment, thefatty acids are present in the therapeutic wound healing composition inan amount from about 10% to about 50%, preferably from about 20% toabout 45%, and more preferably from about 25% to about 40%, by weight ofthe therapeutic wound healing composition.

In accord with the present invention, the therapeutic wound healingcompositions of Embodiment One (I.A-D) for treating mammalian cells maybe selected from the group consisting of:

(I.A)

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.B)

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.C)

(a) an antioxidant; and

(b) a mixture of sainted and unsaturated fatty acids wherein the fattyadds are those fatty acids required for the repair of cellular membranesand resuscitation of mammalian cells;

(I.D)

(a) lactate selected from the group consisting of lactic add,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyadds are those fatty acids required for the repair of cellular membranesand resuscitation of mammalian cells.

Preferably, the wound healing compositions of Embodiment One (I) fortreating mammalian cells, preferably epidermal keratinocytes, may beselected from the group consisting of:

(I.A)

(a) pyruvate selected from the group consisting of pyrovic acid,pharmaceutically acceptable salts of pyrovic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of sainted and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.B)

(a) pyruvate selected from the group consisting ofpyruvic acid,pharmaceutically acceptable salts of pyrovic acid, and mixtures thereof;

(b) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(I.C)

(a) an antioxidant; and

(b) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

More preferably, the wound healing compositions of Embodiment One (I)for treating mammalian cells, preferably epidermal keratinocytes, may beselected from the group consisting of:

(I.A)

(a) pyruvate selected from the group consisting ofpyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(I.C)

(a) an antioxidant; and

(b) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

More preferably, the wound healing compositions of Embodiment One (I)for treating mammalian cells, preferably epidermal keratinocytes, may beselected from the group consisting of:

(I.A)

(a) pyruvate selected from the group consisting ofpyruvic acid,pharmaceutically acceptable salts of pyrovic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(I.B)

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyrovic acid, and mixtures thereof;

(b) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;and

(c) a mixture of sainted and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

Most preferably, the wound healing compositions of Embodiment One (I)for treating mammalian cells, preferably epidermal keratinocytes,comprise:

(I.A)

(a) pyruvate selected from the group consisting ofpyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

Most preferably, the wound healing compositions of Embodiment One (I)for treating mammalian cells, preferably monocytes, comprise:

(I.D)

(a) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

Throughout this disclosure, applicant will suggest various theories ormechanisms by which applicant believes the components in the therapeuticwound healing compositions and the antiviral agent function together inan unexpected synergistic manner to prevent and reduce injury tomammalian cells, increase the resuscitation rate of injured mammaliancells, and reduce viral titers. While applicant may offer variousmechanisms to explain the present invention, applicant does not wish tobe bound by theory. These theories are suggested to better understandthe present invention but are not intended to limit the effective scopeof the claims.

In the first aspect of Embodiment One (I.A), applicant believes thatpyruvate can be transported inside a cell where it can act as anantioxidant to neutralize oxygen radicals in the cell. Pyruvate can alsobe used inside the cell in the citric acid cycle to provide energy toincrease cellular viability, and as a precursor in the synthesis ofimportant biomolecules to promote cellular proliferation. In addition,pyruvate can be used in the multifunction oxidase system to reversecytotoxicity. Antioxidants, especially lipid-soluble antioxidants, canbe absorbed into the cell membrane to neutralize oxygen radicals andthereby protect the membrane. The saturated and unsaturated fatty acidsin the present invention are those fatty acids required for theresuscitation of mammalian cells and are readily available for therepair of injured cells and the proliferation of new cells. Cellsinjured by oxygen radicals need to produce unsaturated fatty acids torepair cellular membranes. However, the production of unsaturated fattyacids by cells requires oxygen. Thus, the injured cell needs high levelsof oxygen to produce unsaturated fatty acids and at the same time needsto reduce the level of oxygen within the cell to reduce oxidativeinjury. By providing the cell with the unsaturated fatty acids neededfor repair, the need of the cell for unsaturated fatty acids is reducedand the need for high oxygen levels is also reduced.

The combination of pyruvate inside the cell and an antioxidant in thecellular membrane functions in an unexpected synergistic manner toreduce hydrogen peroxide production in the cell to levels lower than canbe achieved by use of either type of component alone. The presence ofmixtures of saturated and unsaturated fatty acids in the therapeuticwound healing composition significantly enhances the ability of pyruvateand the antioxidant to inhibit reactive oxygen production. Bystabilizing the cellular membrane, unsaturated fatty acids also improvemembrane function and enhance pyruvate transport into the cell. Hence,the three components in the therapeutic wound healing composition of thefirst aspect of Embodiment One (I.A) function together in an unexpectedsynergistic manner to prevent and reduce injury to mammalian cells andincrease the resuscitation rate of injured mammalian cells.

In the second aspect of Embodiment One (I.B), lactate is employedinstead of an antioxidant. Antioxidants react with, and neutralize,oxygen radicals after the radicals are already formed. Lactate, on theother hand, is a component in the cellular feedback mechanism andinhibits the respiratory bursting process to suppress the production ofactive oxygen species. The combination of pyruvate to neutralize activeoxygen species and lactate to suppress the respiratory bursting processfunctions in a synergistic manner to reduce hydrogen peroxide productionin the cell to levels lower than can be achieved by use of either typeof component alone. The presence of mixtures of saturated andunsaturated fatty acids in the therapeutic wound healing compositionsignificantly enhances the ability ofpyruvate and lactate to inhibitreactive oxygen production. Hence, the three components in thetherapeutic wound healing composition in the second aspect of EmbodimentOne (I.B) function together in a synergistic manner to protect andresuscitate mammalian cells.

In the third aspect of Embodiment One (I.C), the presence of mixtures ofsaturated and unsaturated fatty acids in the therapeutic wound healingcomposition in this embodiment significantly enhances the ability of theantioxidant to inhibit reactive oxygen production. The combination of anantioxidant to neutralize active oxygen species and fatty acids torebuild cellular membranes and reduce the need of the cell for oxygenfunctions in a synergistic manner to reduce hydrogen peroxide productionin the cell to levels lower than can be achieved by either type ofcomponent alone. Hence, the components in the therapeutic wound healingcomposition in the third aspect of Embodiment One (I.C) functiontogether in a synergistic manner to protect and resuscitate mammaliancells.

In the fourth aspect of Embodiment One (I.D), lactate is employedbecause the respiratory bursting process is more pronounced in monocytesthan in epidermal keratinocytes. The combination of lactate to suppressthe respiratory bursting process and an antioxidant to neutralize activeoxygen species functions in a synergistic manner to reduce hydrogenperoxide production in the cell to levels lower than can be achieved byeither component alone. The presence of mixtures of saturated andunsaturated fatty acids in the therapeutic wound healing composition inthis embodiment significantly enhances the ability of lactate and theantioxidant to inhibit reactive oxygen production. Hence, the threecomponents in the therapeutic wound healing composition in the fourthaspect of Embodiment One (I.D) function together in an unexpectedsynergistic manner to protect and resuscitate mammalian cells.

Accordingly, the combination of ingredients set out in the aboveembodiments functions together in an enhanced manner to prevent andreduce injury to mammalian cells and increase the resuscitation rate ofinjured mammalian cells. The therapeutic effect of the combination ofthe components in each of the above embodiments is markedly greater thanthat expected by the mere addition of the individual therapeuticcomponents. Hence, applicant's therapeutic wound healing compositionsfor treating mammalian cells have the ability to decrease intracellularlevels of hydrogen peroxide production, increase cellular resistance tocytotoxic agents, increase rates of cellular proliferation, and increasecellular viability.

B. Methods For Making The Therapeutic Wound Healing Compositions OfEmbodiment One (I.A-D)

The present invention extends to methods for making the therapeuticwound healing compositions of Embodiment One (I.A-D). In general, atherapeutic wound healing composition is made by forming an admixture ofthe components of the composition. In a first aspect of Embodiment One(I.A), a therapeutic wound healing composition is made by forming anadmixture of (a) pyruvate selected from the group consisting of pyruvicacid, pharmaceutically acceptable salts of pyrovic acid, and mixturesthereof, (b) an antioxidant, and (c) a mixture of saturated andunsaturated fatty acids wherein the fatty acids are those fatty acidsrequired for the repair of cellular membranes and resuscitation ofmammalian cells. In a second aspect of Embodiment One (I.B), atherapeutic wound healing composition is made by forming an admixture of(a) pyruvate selected from the group consisting of pyrovic acid,pharmaceutically acceptable salts of pyrovic acid, and mixtures thereof,Co) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof,and (c) a mixture of saturated and unsaturated fatty acids wherein thefatty acids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells. In a third aspect ofEmbodiment One (I.C), a therapeutic wound healing composition is made byforming an admixture of (a) an antioxidant and (b) a mixture ofsaturated and unsaturated fatty acids wherein the fatty acids are thosefatty acids required for the repair of cellular membranes andresuscitation of mammalian cells. In a fourth aspect of Embodiment One(I.D), a therapeutic wound healing composition is made by forming anadmixture of (a) lactate selected from the group consisting of lacticacid, pharmaceutically acceptable salts of lactic acid, and mixturesthereof, (b) an antioxidant, and (c) a mixture of saturated andunsaturated fatty acids wherein the fatty acids are those fatty acidsrequired for the repair of cellular membranes and resuscitation ofmammalian cells.

For some applications, the admixture may be formed in a solvent such aswater, and a surfactant may be added if required. If necessary, the pHof the solvent is adjusted to a range from about 3.5 to about 8.0, andpreferably from about 4.5 to about 7.5, and more preferably about 6.0 toabout 7.4. The admixture is then sterile filtered. Other ingredients mayalso be incorporated into the therapeutic wound healing composition asdictated by the nature of the desired composition as well known by thosehaving ordinary skill in the art. The ultimate therapeutic wound healingcompositions are readily prepared using methods generally known in thepharmaceutical arts.

In a preferred embodiment, the invention is directed to a method forpreparing a therapeutic wound healing composition (I.A) for preventingand reducing injury to mammalian cells, and increasing the resuscitationrate of injured mammalian cells, which comprises the steps of admixingthe following ingredients:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyrovic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the resuscitation of injuredmammalian cells.

C. Methods For Employing The Therapeutic Wound Healing Compositions OfEmbodiment One (I.A-D)

The present invention extends to methods for employing the therapeuticwound healing compositions of Embodiment One (I) in vivo and in vitro.In general, a therapeutic wound healing composition is employed bycontacting the therapeutic composition with mammalian cells.

In a first aspect of Embodiment One (I.A), the invention is directed toa method for preventing and reducing injury to mammalian cells, andincreasing the resuscitation rate of injured mammalian cells, whichcomprises the steps of (A) providing a therapeutic wound healingcomposition which comprises (a) pyruvate selected from the groupconsisting of pyrovic acid, pharmaceutically acceptable salts of pyruvicacid, and mixtures thereof, (b) an antioxidant, and (c) a mixture ofsaturated and unsaturated fatty acids wherein the fatty acids are thosefatty acids required for the resuscitation of injured mammalian cells,and (B) contacting the therapeutic wound healing composition with themammalian cells.

In a second aspect of Embodiment One (I.B), the invention is directed toa method for preventing and reducing injury to mammalian cells, andincreasing the resuscitation rate of injured mammalian cells, whichcomprises the steps of (A) providing a therapeutic wound healingcomposition which comprises (a) pyruvate selected from the groupconsisting of pyrovic acid, pharmaceutically acceptable salts of pyruvicacid, and mixtures thereof, (b) lactate selected from the groupconsisting of lactic acid, pharmaceutically acceptable salts of lacticacid, and mixtures thereof, and (c) a mixture of saturated andunsaturated fatty acids wherein the fatty acids are those fatty acidsrequired for the resuscitation of injured mammalian cells, and (B)contacting the therapeutic wound healing composition with the mammaliancells.

In a third aspect of Embodiment One (I.C), the invention is directed toa method for preventing and reducing injury to mammalian cells, andincreasing the resuscitation rate of injured mammalian cells, whichcomprises the steps of (A) providing a therapeutic wound healingcomposition which comprises (a) an antioxidant, and (b) a mixture ofsaturated and unsaturated fatty acids wherein the fatty acids are thosefatty acids required for the resuscitation of injured mammalian cells,and (B) contacting the therapeutic wound healing composition with themammalian cells.

In a fourth aspect of Embodiment One (I.D), the invention is directed toa method for preventing and reducing injury to mammalian cells, andincreasing the resuscitation rate of injured mammalian cells, whichcomprises the steps of (A) providing a therapeutic wound healingcomposition which comprises (a) lactate selected from the groupconsisting of lactic acid, pharmaceutically acceptable salts of lacticacid, and mixtures thereof, (b) an antioxidant, and (c) a mixture ofsaturated and unsaturated fatty acids wherein the fatty acids are thosefatty acids required for the resuscitation of injured mammalian cells,and (B) contacting the therapeutic wound healing composition with themammalian cells.

In a preferred embodiment, the invention is directed to a method forhealing a wound in a mammal which comprises the steps of:

(A) providing a therapeutic wound healing composition (I.A) whichcomprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the resuscitation of injuredmammalian cells; and

(B) contacting the therapeutic wound healing composition with the wound.

The types of wounds which may be healed using the wound healingcompositions of Embodiment One (I.A-D) of the present invention arethose which result from an injury which causes epidermal damage such asincisions, wounds in which the skin is broken by a cutting instrument,and lacerations, wounds in which the skin is broken by a dull or bluntinstrument. The therapeutic compositions may also be used to treatvarious dermatological disorders such as hyperkeratosis, photo-aging,burns, donor site wounds from skin transplants, ulcers (cutaneous,decubitus, venous stasis, and diabetic), psoriasis, skin rashes, andsunburn photoreactive processes. The topical therapeutic compositionsmay also be used orally in the form of a mouth wash or spray to protectand accelerate the healing of injured oral tissue such as mouth soresand burns. The topical therapeutic compositions may further be used inophthalmological preparations to treat wounds such as those which resultfrom corneal ulcers, radialkeratotomy, corneal transplants,epikeratophakia and other surgically induced wounds in the eye. Thetopical therapeutic compositions may in addition be used in anorectalcreams and suppositories to treat such conditions as pruritus and,proctitis, anal fissures, and hemorrhoids. In a preferred embodiment,the therapeutic compositions are used to treat wounds such as incisionsand lacerations.

The wound healing compositions of Embodiment One (I.A-D) of the presentinvention may be utilized in topical products, ingestible products, andtissue culture medium to protect mammalian cells and increase theresuscitation rate of injured mammalian cells. For example, thetherapeutic wound healing compositions may be used in topical skin careproducts to protect and increase the resuscitation rate of skin tissuesuch as in the treatment of various dermatological disorders such ashyperkeratosis, photo-aging, and sunburn photoreactive processes. Injuryto skin can occur for a variety of reasons. Injury often occurs toindividuals who wash their hands often, to individuals who are exposedto stressful environmental conditions (overexposure to sun orchemicals), or to the elderly or individuals with an underliningdisease. The addition of the wound healing compositions of the presentinvention to a lotion provides a source of antioxidants to the skinwhich would protect the skin from the harmful effects of UV light,chemicals, and severe drying. The wound healing compositions can be usedfor the following indications: a) Moisturizing and protecting; b)Healing dry cracked skin; c) Treating irritated skin such as diaperrash; d) Healing severe dry skin due to other diseases (venousdermatitis); e) Treating psoriasis and other hyperproliferativediseases; f) Protecting skin from UV light damage (antioxidant skinreplacement); g) Treating seborrheic conditions; and h) Treating shavingwounds in an after shave lotion.

The topical therapeutic wound healing compositions may also be usedorally in the form of a mouth wash or spray to protect and acceleratethe healing of injured oral tissue such as mouth sores and bums. Thetopical therapeutic wound healing compositions may further be used inophthalmological preparations such as eye care products to neutralizehydrogen peroxide used in the cleaning of contact lenses. The topicaltherapeutic wound healing compositions may in addition be used inanorectal creams and suppositories to treat such conditions as pruritusand, proctitis, anal fissures, and hemorrhoids. Initially as white bloodcells enter a wound site, the cells release oxygen radicals, depletingthe andoxidants at the wound site, thus impairing the healing process.Incorporating the wound healing compositions of the present inventioninto a wound healing formulation would facilitate healing by providingthe site with usable antioxidants, and a source of fatty acids neededfor membrane repair. The wound healing compositions can be used for thefollowing indications: a) Healing of cuts and scrapes; b) Burns (healsburns with less scaring and scabbing); c) Decubitus ulcers; d) Bedsores, pressure ulcers; e) Fissures, Hemorrhoids; f) Use in combinationwith immunostimulators (simulated healing in healing deficient people);g) Post surgical wounds; h) Bandages; i) Diabetic ulcers; j) Venousulceration; and k) Use in combination with wound cleansing agents.

The therapeutic wound healing compositions may also be used iningestible products to protect and increase the resuscitation rate oferosions, stomach ulcers, and hemorrhages in the gastric mucosa. Otheringestible therapeutic products include: stroke medications; autoimmunedisease medications; arthritis medications; ulcer medications; cancermedications (cytotoxic agents); heart medication to improve regionalventricular function and restore normal heart rate and pressurefunctions; lung medication to repair injured tissue; liver medication tosuppress lipogenesis of alcoholic origin and prevent hepatic steatosis;kidney medication to suppress urinary calculi (kidney stones);detoxification medication to antagonize heavy metal poisoning, cyanidepoisoning, sodium sulfide poisoning, other types of poisoning, andreduce and neutralize the production of oxygen radicals which producesinjury to tissue, to protect and further enhance the resuscitation rateof the injured mammalian cells. The therapeutic wound healingcompositions may be used in ingestible products to treat inflammatorydiseases such as hepatitis, gastritis, colitis, esophagitis, arthritis,and pancreatitis.

The therapeutic wound healing compositions of the present invention mayalso be used in tissue culture media and organ transplant media toprevent and reduce injury to mammalian cells and increase theresuscitation rate of injured mammalian cells. Tissue cultures andtransplant organs encounter reactive oxygen species generated in theculture media by the injured cells. Organs particularly susceptible tooxidative damage during transport and transplantation due to reperfusioninjury following ischemia are comeas, livers, hearts, and kidneys. Thetherapeutic wound healing compositions may be usefid to abrogatereperfusion injury to such transplant organs.

In a specific embodiment, the invention is directed to a method forpreserving mammalian cells in a culture medium which comprises the stepsof:

(A) providing a therapeutic wound healing composition selected from thegroup of consisting of:

(I.A)

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.B)

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.C)

(a) an antioxidant; and

(b) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.D)

(a) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the resuscitation of injuredmammalian cells;

(B) providing mammalian cells in a culture medium; and

(C) contacling the therapeutic wound healing composition from step (A)with the mammalian cells in the culture medium from step (B).

D. Formulations Of The Therapeutic Wound Healing Compositions OfEmbodiment One (I.A-D)

Once prepared, the inventive therapeutic wound healing compositions ofEmbodiment One (I.A-D) may be stored for future use or may be formulatedin effective amounts with pharmaceutically acceptable carriers toprepare a wide variety of pharmaceutical compositions. Examples ofpharmaceutically acceptable carriers are pharmaceutical appliances,topical vehicles (non-oral and oral), and ingestible vehicles.

Examples of pharmaceutical appliances are sutures, staples, gauze,bandages, burn dressings, artificial skins, liposome or micellformulations, microcapsules, aqueous vehicles for soaking gauzedressings, and the like, and mixtures thereof. Non-oral topicalcompositions employ non-oral topical vehicles, such as creams, gelsformulations, foams, ointments and sprays, salves, and films, which areintended to be applied to the skin or body cavity and are not intendedto be taken by mouth. Oral topical compositions employ oral vehicles,such as mouthwashes, rinses, oral sprays, suspensions, and dental gels,which are intended to be taken by mouth but are not intended to beingested. Ingestible compositions employ ingestible or partly ingestiblevehicles such as confectionery bulking agents which include hard andsoft confectionery such as lozenges, tablets, toffees, nougats,suspensions, chewy candies, and chewing gums.

In one form of the invention, the therapeutic wound healing compositionis incorporated into a pharmaceutical appliance which may be in the formof sutures, staples, gauze, bandages, burn dressings, artificial skins,liposome or micell formulations, microcapsules, aqueous vehicles forsoaking gauze dressings, and the like, and mixtures thereof. A varietyof traditional ingredients may optionally be included in thepharmaceutical composition in effective amounts such as buffers,preservatives, tonicity adjusting agents, antioxidants, polymers foradjusting viscosity or for use as extenders, and excipients, and thelike. Specific illustrative examples of such traditional ingredientsinclude acetate and borate buffers; thimerosal, sorbic acid, methyl andpropyl paraben and chlorobutanol preservatives; sodium chloride andsugars to adjust the tonicity; and excipients such as marmitol, lactoseand sucrose. Other conventional pharmaceutical additives known to thosehaving ordinary skill in the pharmaceutical arts may also be used in thepharmaceutical composition.

In accordance with this invention, therapeutically effective amounts ofthe therapeutic wound healing compositions of the present invention maybe employed in the pharmaceutical appliance. These amounts are readilydetermined by those skilled in the an without the need for undueexperimentation. The exact amount of the therapeutic wound healingcomposition employed is subject to such factors as the type andconcentration of the therapeutic wound healing composition and the typeof pharmaceutical appliance employed. Thus, the amount of therapeuticwound healing composition may be varied in order to obtain the resultdesired in the final product and such variations are within thecapabilities of those skilled in the an without the need for undueexperimentation. In a preferred embodiment, the pharmaceuticalcomposition will comprise the therapeutic wound healing composition inan amount from about 0.1% to about 5%, by weight of the pharmaceuticalcomposition. In a more preferred embodiment, the pharmaceuticalcomposition will comprise the therapeutic wound healing composition inan amount from about 0.1% to about 3%, by weight of the pharmaceuticalcomposition. In a most preferred embodiment, the pharmaceuticalcomposition will comprise the therapeutic wound healing composition inan amount from about 0.1% to about 1%, by weight of the pharmaceuticalcomposition.

The present invention extends to methods for making the pharmaceuticalcompositions. In general, a pharmaceutical composition is made bycontacting a therapeutically effective amount of a therapeutic woundhealing composition with a pharmaceutical appliance and the otheringredients of the final desired pharmaceutical composition. Thetherapeutic wound healing composition may be in a solvent and may beabsorbed onto a pharmaceutical appliance.

Other ingredients will usually be incorporated into the composition asdictated by the nature of the desired composition as well known by thosehaving ordinary skill in the art. The ultimate pharmaceuticalcompositions are readily prepared using methods generally known in thepharmaceutical arts.

In another form of the invention, the therapeutic wound healingcomposition is incorporated into a non-oral topical vehicle which may bein the form of a cream, gel, foam, ointment, spray, and the like.Typical non-toxic non-oral topical vehicles known in the pharmaceuticalarts may be used in the present invention. The preferred non-oraltopical vehicles are water and pharmaceutically acceptablewater-miscible organic solvents such as ethyl alcohol, isopropylalcohol, propylene glycol, glycerin, and the like, and mixtures of thesesolvents. Water-alcohol mixtures are particularly preferred and aregenerally employed in a weight ratio from about 1:1 to about 20:1,preferably from about 3:1 to about 20:1, and most preferably from about3:1 to about 10:1, respectively.

The non-oral topical therapeutic wound healing compositions may alsocontain conventional additives employed in those products. Conventionaladditives include humectants, emollients, lubricants, stabilizers, dyes,and perfumes, providing the additives do not interfere with thetherapeutic properties of the therapeutic wound healing composition.

Suitable humectants useful in the non-oral topical therapeutic woundhealing compositions include glycerin, propylene glycol, polyethyleneglycol, sorbitan, fructose, and the like, and mixtures thereof.Humectants, when employed, may be present in amounts from about 10% toabout 20%, by weight of the topical therapeutic wound healingcomposition.

The coloring agents (colors, colorants) useful in the non-oral topicaltherapeutic wound healing composition are used in amounts effective toproduce the desired color. These coloring agents include pigments whichmay be incorporated in amounts up to about 6% by weight of the non-oraltopical therapeutic wound healing composition. A preferred pigment,titanium dioxide, may be incorporated in amounts up to about 2%, andpreferably less than about 1%, by weight of the non-oral topicaltherapeutic wound healing composition. The coloring agents may alsoinclude natural food colors and dyes suitable for food, drug andcosmetic applications. These coloring agents are known as F.D.& C. dyesand lakes. The materials acceptable for the foregoing uses arepreferably water-soluble. Illustrative nonlimiting examples include theindigoid dye known as F.D.& C. Blue No.2, which is the disodium salt of5,5-indigotindisulfonic acid. Similarly, the dye known as F.D.& C. GreenNo.1 comprises a triphenylmethane dye and is the monosodium salt of4-[4-(N-ethyl-p-sulfoniumbenzylamino)diphenylmethylene]-[1-(N-ethyl-N-p-sulfoniumbenzyl)-delta-2,5-cyclohexadieneimine].A full recitation of all F.D.& C. coloring agents and theircorresponding chemical structures may be found in the Kirk-OthmerEncyclopedia of Chemical Technology, 3rd Edition, in volume 5 at pages857-884, which text is incorporated herein by reference.

In accordance with this invention, therapeutically effective amounts ofthe therapeutic wound healing compositions of the present invention maybe admixed with a non-oral topical vehicle to form a topical therapeuticwound healing composition. These amounts are readily determined by thoseskilled in the art without the need for undue experimentation. In apreferred embodiment, the non-oral topical therapeutic wound healingcompositions will comprise the therapeutic wound healing composition inan amount from about 0.1% to about 10% and a non-oral topical vehicle ina quantity sufficient to bring the total amount of composition to 100%,by weight of the non-oral topical therapeutic wound healing composition.In a more preferred embodiment, the non-oral topical therapeutic woundhealing compositions will comprise the therapeutic wound healingcomposition in an amount from about 0.1% to about 5%, and in a mostpreferred embodiment, the non-oral topical therapeutic wound healingcompositions will comprise the therapeutic wound healing composition inan amount from about 0.1% to about 2%, and a non-oral topical vehicle ina quantity sufficient to bring the total amount of composition to 100%,by weight of the non-oral topical therapeutic wound healing composition.

The present invention extends to methods for preparing the non-oraltopical therapeutic wound healing compositions. In such a method, thenon-oral topical therapeutic wound healing composition is prepared byadmixing a therapeutically effective amount of the therapeutic woundhealing composition of the present invention and a non-oral topicalvehicle. The final compositions are readily prepared using standardmethods and apparatus generally known by those skilled in thepharmaceutical arts. The apparatus useful in accordance with the presentinvention comprises mixing apparatus well known in the pharmaceuticalarts, and therefore the selection of the specific apparatus will beapparent to the artisan.

In another form of the invention, the therapeutic wound healingcomposition is incorporated into an oral topical vehicle which may be inthe form of a mouthwash, rinse, oral spray, suspension, dental gel, andthe like. Typical non-toxic oral vehicles known in the pharmaceuticalarts may be used in the present invention. The preferred oral vehiclesare water, ethanol, and water-ethanol mixtures. The waterethanolmixtures are generally employed in a weight ratio from about 1:1 toabout 20:1, preferably from about 3:1 to about 20:1, and most preferablyfrom about 3:1 to about 10:1, respectively. The pH value of the oralvehicle is generally from about 4 to about 7, and preferably from about5 to about 6.5. An oral topical vehicle having a pH value below about 4is generally irritating to the oral cavity and an oral vehicle having apH value greater than about 7 generally results in an unpleasant mouthfeel.

The oral topical therapeutic wound healing compositions may also containconventional additives normally employed in those products. Conventionaladditives include a fluorine providing compound, a sweetening agent, aflavoring agent, a coloring agent, a humectant, a buffer, and anemulsifier, providing the additives do not interfere with thetherapeutic properties of the therapeutic wound healing composition.

The coloring agents and humectants, and the amounts of these additivesto be employed, set out above as useful in the non-oral topicaltherapeutic wound healing composition may be used in the oral topicaltherapeutic wound healing composition.

Fluorine providing compounds may be fully or slightly water soluble andare characterized by their ability to release fluoride ions or fluoridecontaining ions in water and by their lack of reaction with othercomponents in the composition. Typical fluorine providing compounds areinorganic fluoride salts such as water-soluble alkali metal, alkalineearth metal, and heavy metal salts, for example, sodium fluoride,potassium fluoride, ammonium fluoride, cuprous fluoride, zinc fluoride,stannic fluoride, stannous fluoride, barium fluoride, sodiumfluorosilicate, ammonium fluorosilicate, sodium fluorozirconate, sodiummonofluorophosphate, aluminum mono- and di-fluorophosphates andfluorinated sodium calcium pyrophosphate. Alkali metal fluorides, tinfluoride and monofluorophosphates, such as sodium and stannous fluoride,sodium monofluorophosphate and mixtures thereof, are preferred.

The amount of fluorine providing compound present in the present oraltopical therapeutic wound healing composition is dependent upon the typeof fluorine providing compound employed, the solubility of the fluorinecompound, and the nature of the final oral therapeutic wound healingcomposition. The amount of fluorine providing compound used must be anontoxic amount. In general, the fluorine providing compound when usedwill be present in an amount up to about 1%, preferably from about0.001% to about 0.1%, and most preferably from about 0.001% to about0.05%, by weight of the oral topical therapeutic wound healingcomposition.

When sweetening agents (sweeteners) are used, those sweeteners wellknown in the art, including both natural and artificial sweeteners, maybe employed. The sweetening agent used may be selected from a wide rangeof materials including water-soluble sweetening agents, water-solubleartificial sweetening agents, water-soluble sweetening agents derivedfrom naturally occurring water-soluble sweetening agents, dipeptidebased sweetening agents, and protein based sweetening agents, includingmixtures thereof. Without being limited to particular sweetening agents,representative categories and examples include:

(a) water-soluble sweetening agents such as monosaccharides,disaccharides and polysaccharides such as xylose, ribose, glucose(dextrose), mannose, galactose, fructose (levulose), sucrose (sugar),maltose, invert sugar (a mixture of fruclose and glucose derived fromsucrose), partially hydrolyzed starch, corn syrup solids,dihydrochalcones, monellin, steviosides, and glycyrrhizin, and mixturesthereof;

(b) water-soluble artificial sweeteners such as soluble saccharin salts,i.e., sodium or calcium saccharin salts, cyclamate salts, the sodium,ammonim or calcium salt of3,4-dihydro-6-methyl-1,2,3-oxathiazine-4-one-2,2-dioxide, the potassiumsalt of 3,4-dihydro-6-methyl-1,2,3-oxathiazine-4-one-2,2-dioxide(Acesulfame-K), the free acid form of saccharin, and the like;

(c) dipeptide based sweeteners, such as L-aspartic acid derivedsweeteners, such as L-aspartyl-L-phenylalanine methyl ester (Aspartame)and materials described in U.S. Pat. No. 3,492,131,L-Alpha-aspartyl-N-(2,2,4,4-tetramethyl-3-thietanyl)-D-alanin-amidehydrate (Alitame), methyl esters of L-aspartyl-L-phenylglycerineandL-aspartyl-L-2,5-dihydrophenyl-glycine,L-aspartyl-2,5-dihydro-L-phenylalanine;L-aspartyl-L-(1-cyclohexen)-alanine, and the like;

(d) water-soluble sweeteners derived from naturally occurringwater-soluble sweeteners, such as chlorinated derivatives of ordinarysugar (sucrose), e.g., chlorodeoxysugar derivatives such as derivativesof chlorodeoxysucrose or chlorodeoxygalactosucrose, known, for example,under the product designation of Sucralose; examplesofchlorodeoxysucrose and chlorodeoxygalacto-sucrose derivatives includebut are not limited to: 1-chloro-1'-deoxysucrose;4-chloro-4-deoxy-Alpha-D-galacto-pyranosyl-Alpha-D-fructofuranoside, or4-chloro-4-deoxygalactosucrose;4-chloro-4-deoxy-Alpha-D-galacto-pyranosyl-1-chloro-1-deoxy-β-D-fruto-furanoside,or 4,1'-dichloro-4,1'-dideoxygalactosucrose;1',6'-dichloro-1',6'-dideoxysucrose;4-chloro-4-deoxy-Alpha-D-galacto-pyranosyl-1,6-dichloro-1,6-dideoxy-β-D-fructo-furanoside,or 4,1',6'-trichloro-4,1',6'-trideoxygalacto-sucrose;4,6-dichloro-4,6-dideoxy-Alpha-D-galacto-pyranosyl-6-chloro-6-deoxy-β-D-fructofumnoside,or 4,6,6'-trichloro-4,6,6'-trideoxygalactosucrose;6,1',6'-trichloro-6,1',6'-trideoxysucrose;4,6-dichloro-4,6-dideoxy-Alpha-D-galacto-pyranosyl-1,6-dichloro-1,6-di-deoxy-β-D-fructofuranoside,or 4,6,1',6'-tetrachloro-4,6,1',6'-tetradeoxygalacto-sucrose; and4,6,1',6'-tetrachloro-4,6,1',6'-tetradeoxy-sucrose; and

(e) protein based sweeteners such as thaumaoccous danielli (Thaumatin Iand lI).

In general, an effective amount of sweetening agent is utilized toprovide the level of sweetness desired in the particular oral topicaltherapeutic wound healing composition, and this amount will vary withthe sweetener selected and the final oral therapeutic product desired.The amount of sweetener normally present is in the range from about0.0025% to about 90%, by weight of the oral topical therapeutic woundhealing composition, depending upon the sweetener used. The exact rangeof amounts for each type of sweetener is well known in the art and isnot the subject of the present invention.

The flavoring agents (flavors, flavorants) which may be used includethose flavors known to the skilled artisan, such as natural andartificial flavors. Suitable flavoring agents include mints, such aspeppermint, citrus flavors such as orange and lemon, artificial vanilla,cinnamon, various fruit flavors, both individual and mixed, and thelike.

The amount of flavoring agent employed in the oral topical therapeuticwound healing composition is normally a matter of preference subject tosuch factors as the type of final oral therapeutic wound healingcomposition, the individual flavor employed, and the strength of flavordesired. Thus, the amount of flavoring may be varied in order to obtainthe result desired in the final product and such variations are withinthe capabilities of those skilled in the art without the need for undueexperimentation. The flavoring agents, when used, are generally utilizedin amounts that may, for example, range in amounts from about 0.05% toabout 6%, by weight of the oral topical therapeutic wound healingcomposition.

Suitable buffer solutions useful in the non-oral topical therapeuticwound healing compositions include citric acid-sodium citrate solution,phosphoric acid-sodium phosphate solution, and acetic acid-sodiumacetate solution in amounts up to about 1%, and preferably from about0.05% to about 0.5% by weight of the oral topical therapeutic woundhealing composition.

In accordance with this invention, therapeutically effective amounts ofthe therapeutic wound healing compositions of the present invention maybe admixed with an oral topical vehicle to form a topical therapeuticwound healing composition. These amounts are readily determined by thoseskilled in the art without the need for undue experimentation. In apreferred embodiment, the oral topical therapeutic wound healingcompositions will comprise the therapeutic wound healing composition inan amount from about 0.1% to about 10% and a oral topical vehicle in aquantity sufficient to bring the total amount of composition to 100%, byweight of the oral topical therapeutic wound healing composition. In amore preferred embodiment, the oral topical therapeutic wound healingcompositions will comprise the therapeutic wound healing composition inan amount from about 0.1% to about 5%, and in a most preferredembodiment, the oral topical therapeutic wound healing compositions willcomprise the therapeutic wound healing composition in an amount fromabout 0.1% to about 2%, and a oral topical vehicle in a quantitysufficient to bring the total amount of composition to 100%, by weightof the oral topical therapeutic wound healing composition.

The present invention extends to methods for preparing the oral topicaltherapeutic wound healing compositions. In such a method, the oraltopical therapeutic wound healing composition is prepared by admixing atherapeutically effective amount of the therapeutic wound healingcomposition of the present invention and an oral topical vehicle. Thefinal compositions are readily prepared using standard methods andapparatus generally known by those sldlled in the pharmaceutical arts.The apparatus useful in accordance with the present invention comprisesmixing apparatus well known in the pharmaceutical arts, and thereforethe selection of the specific apparatus will be apparent to the artisan.

In a preferred embodiment, an oral topical therapeutic wound healingcomposition is made by first dissolving coloring agents, sweeteningagents, and similar additives in water. The therapeutic wound healingcomposition is then admixed with the aqueous solution. Then sufficientwater or ethanol, or mixtures of water and ethanol, are added to thesolution with mixing until the final solution volume is reached. In amore preferred embodiment, the therapeutic wound healing composition isadded to the solution as the final ingredient. The final oral topicaltherapeutic wound healing compositions are readily prepared usingmethods generally known in the pharmaceutical arts.

The oral therapeutic wound healing composition may also be in the formof dental gel. As used herein, the term "gel" means a solid or semisolidcolloid which contains considerable quantities of water. The colloidparticles in a gel are linked together in a coherent meshwork whichimmobflizes the water contained inside the meshwork.

The dental gel compositions of the present invention may contain theconventional additives set out above for oral topical therapeutic woundhealing compositions such as mouthwashes, rinses, oral sprays, andsuspensions and, in addition, may contain additional additives such as apolishing agent, a desensitizing agent, and the like, providing theadditional additives do not interfere with the therapeutic properties ofthe therapeutic wound healing composition.

In a dental gel composition, the oral vehicle generally comprises water,typically in an amount from about 10% to about 90%, by weight of thedental gel composition. Polyethylene glycol, propylene glycol, glycerin,and mixtures thereof may also be present in the vehicle as humectants orbinders in amounts from about 18% to about 30%, by weight of the dentalgel composition. Particularly preferred oral vehicles comprise mixturesof water with polyethylene glycol or water with glycerin andpolypropylene glycol.

The dental gels of the present invention include a gelling agent(thickening agent) such as a natural or synthetic gum or gelatin.Gelling agents such as hydroxyethyl cellulose, methyl cellulose,glycerin, carboxypolymethylene, and gelatin and the like, and mixturesthereof may be used. The preferred gelling agent is hydroxyethylcellulose. Gelling agents may be used in amounts from about 0.5% toabout 5%, and preferably from about 0.5% to about 2%, by weight of thedental gel composition.

The dental gel compositions of the present invention may also include apolishing agent. In clear gels, a polishing agent of colloidal silicaand/or alkali metal aluminosilicate complexes is preferred since thesematerials have refractive indices close to the refractive indices of thegelling systems commonly used in dental gels. In non-clear gels, apolishing agent of calcium carbonate or calcium dihydrate may be used.These polishing agents may be used in amounts up to about 75%, andpreferably in amounts up to about 50%, by weight of the dental gelcomposition.

The dental gel may also contain a desensitizing agent such as acombination of citric acid and sodium citrate. Citric acid may be usedin an amount from about 0.1% to about 3%, and preferably from about 0.2%to about 1%, by weight, and sodium citrate may be used in an amount fromabout 0.3% to about 9%, and preferably from about 0.6% to about 3%, byweight of the dental gel composition.

In accordance with this invention, therapeutically effective amounts ofthe therapeutic wound healing compositions of the present invention maybe admixed into the dental gel compositions. These amounts are readilydetermined by those skilled in the art without the need for undueexperimentation. In a preferred embodiment, the dental gel compositionswill comprise the therapeutic wound healing composition in an amountfrom about 0.1% to about 10% and an oral topical vehicle in a quantitysufficient to bring the total amount of composition to 100%, by weightof the dental gel composition. In a more preferred embodiment, thedental gel compositions will comprise the therapeutic wound healingcomposition in an amount from about 0.1% to about 5%, and in a mostpreferred embodiment, the dental gel compositions will comprise thetherapeutic wound healing composition in an amount from about 0.1% toabout 2%, and an oral topical vehicle in a quantity sufficient to bringthe total amount of composition to 100%, by weight of the dental gelcomposition.

The present invention extends to methods for preparing the therapeuticdental gel compositions. In such a method, the dental gel composition isprepared by admixing a therapeutically effective amount of thetherapeutic wound healing composition of the present invention and anoral topical vehicle. The final compositions are readily prepared usingmethods generally known by those skilled in the dental andpharmaceutical arts. The apparatus useful in accordance with the presentinvention comprises mixing apparatus well known in the pharmaceuticalarts, and therefore the selection of the specific apparatus will beapparent to the artisan.

In a preferred embodiment, a therapeutic dental gel composition is madeby first dispersing a gelling agent in a humectant or water, or amixture of both, then admixing to the dispersion an aqueous solution ofthe water-soluble additives such as the fluorine providing compound,sweeteners and the like, then adding the polishing agent, and lastlyadmixing the flavoring agent and the therapeutic wound healingcomposition. The final gel mixture is then tubed or otherwise packaged.The liquids and solids in a gel product are proportioned to form acreamy or gelled mass which is extrudable from a pressurized containeror from a collapsible tube. The final therapeutic wound healingcompositions are readily prepared using methods generally known in thepharmaceutical arts.

In yet another form of the invention, the therapeutic wound healingcomposition is incorporated into an ingestible vehicle. The ingestiblevehicle may be a confectionery bulking agent in the form of lozenges,tablets, toffees, nougats, suspensions, chewy candies, chewing gums, andthe like. The pharmaceutically acceptable carriers may be prepared froma wide range of materials including, but not limited to, diluents,binders and adhesives, lubricants, disintegrants, coloring agents,bulking agents, flavoring agents, sweetening agents and miscellaneousmaterials such as buffers and adsorbents that may be needed in order toprepare a particular therapeutic confection.

The preparation of confectionery formulations is historically well knownand has changed little through the years. Confectionery items have beenclassified as either "hard" confectionery or "soft" confectionery. Thetherapeutic wound healing compositions of the present invention can beincorporated into confectionery compositions by admixing the inventivecomposition into conventional hard and soft confections.

As used herein, the term confectionery material means a productcontaining a bulking agent selected from a wide variety of materialssuch as sugar, corn syrup, and in the case of sugarless bulking agents,sugar alcohols such as sorbitol and mannitol and mixtures thereof.Confectionery material may include such exemplary substances aslozenges, tablets, toffee, nougat, suspensions, chewy candy, chewing gumand the like. The bulking agent is present in a quantity sufficient tobring the total amount of composition to 100%. In general, the bulkingagent will be present in amounts up to about 99.98%, preferably inamounts up to about 99.9%, and more preferably in amounts up to about99%, by weight of the ingestible therapeutic wound healing composition.

Lozenges are flavored medicated dosage forms intended to be sucked andheld in the mouth. Lozenges may be in the form of various shapes such asflat, circular, octagonal and biconvex forms. The lozenge bases aregenerally in two forms: hard boiled candy lozenges and compressed tabletlozenges.

Hard boiled candy lozenges may be processed and formulated byconventional means. In general, a hard boiled candy lozenge has a basecomposed of a mixture of sugar and other carbohydrate bulking agentskept in an amorphous or glassy condition. This amorphous or glassy formis considered a solid syrup of sugars generally having from about 0.5%to about 1.5% moisture. Such materials normally contain up to about 92%corn syrup, up to about 55% sugar and from about 0.1% to about 5% water,by weight of the final composition. The syrup component is generallyprepared from corn syrups high in fmctose, but may include othermaterials. Further ingredients such as flavoring agents, sweeteningagents, acidulants, coloring agents and the like may also be added.

Boiled candy lozenges may also be prepared from non-fermentable sugarssuch as sorbitol, marmitol, and hydrogenated corn syrup. Typicalhydrogenated corn syrups are Lycasin, a commercially available productmanufactured by Roquette Corporation, and Hystar, a commerciallyavailable product manufactured by Lonza, Inc. The candy lozenges maycontain up to about 95% sorbitol, a mixture of sorbitol and mannitol ina ratio from about 9.5:0.5 up to about 7.5:2.5, and hydrogenated cornsyrup up to about 55%, by weight of the solid syrup component.

Boiled candy lozenges may be routinely prepared by conventional methodssuch as those involving fire cookers, vacuum cookers, andscraped-surface cookers also referred to as high speed atmosphericcookers.

Fire cookers involve the traditional method of making a boiled candylozenge base. In this method, the desired quantity of carbohydrate bigagent is dissolved in water by heating the agent in a kettle until thebulking agent dissolves. Additional bulking agent may then be added andcooking continued until a final temperature of 145° C. to 156° C. isachieved. The batch is then cooled and worked as a plastic-like mass toincorporate additives such as flavors, colorants and the like.

A high-speed atmospheric cooker uses a heat-exchanger surface whichinvolves spreading a film of candy on a heat exchange surface, the candyis heated to 165° C. to 170° C. in a few minutes. The candy is thenrapidly cooled to 100° C. to 120° C. and worked as a plastic-like massenabling incorporation of the additives, such as flavors, colorants andthe like.

In vacuum cookers, the carbohydrate bulking agent is boiled to 125° C.to 132° C., vacuum is applied and additional water is boiled off withoutextra heating. When cooking is complete, the mass is a semi-solid andhas a plastic-like consistency. At this point, flavors, colorants, andother additives are admixed in the mass by routine mechanical mixingoperations.

The optimum mixing required to uniformly mix the flavoring agents,coloring agents and other additives during conventional manufacturing ofboiled candy lozenges is determined by the time needed to obtain auniform distribution of the materials. Normally, mixing times of from 4to 10 minutes have been found to be acceptable.

Once the boiled candy lozenge has been properly tempered, it may be cutinto workable portions or formed into desired shapes. A variety offorming techniques may be utilized depending upon the shape and size ofthe final product desired. A general discussion of the composition andpreparation of hard confections may be found in H. A. Lieberman,Pharmaceutical Dosage Forms: Tablets, Volume I (1980), Marcel Dekker,Inc., New York, N.Y. at pages 339 to 469, which disclosure isincorporated herein by reference.

The apparatus useful in accordance with the present invention comprisescooking and mixing apparatus well known in the confectionerymanufacturing arts, and therefore the selection of the specificapparatus will be apparent to the artisan.

In contrast, compressed tablet confections contain particulate materialsand are formed into structures under pressure. These confectionsgenerally contain sugars in amounts up to about 95%, by weight of thecomposition, and typical tablet excipients such as binders andlubricants as well as flavoring agents, coloring agents and the like.

In addition to hard confectionery materials, the lozenges of the presentinvention may be made of soft confectionery materials such as thosecontained in nougat. The preparation of soft confections, such asnougat, involves conventional methods, such as the combination of twoprimary components, namely (1) a high boiling syrup such as a cornsyrup, hydrogenated starch hydrolysate or the like, and (2) a relativelylight textured frappe, generally prepared from egg albumin, gelatin,vegetable proteins, such as soy derived compounds, sugarless milkderived compounds such as milk proteins, and mixtures thereof. Thefrappe is generally relatively light, and may, for example, range indensity from about 0.5 to about 0.7 grams/cc.

The high boiling syrup, or "bob syrup" of the soft confectionery isrelatively viscous and has a higher density than the frappe component,and frequently contains a substantial amount of carbohydrate bulkingagent such as a hydrogenated starch hydrolysate. Conventionally, thefinal nougat composition is prepared by the addition of the "bob syrup"to the frappe under agitation, to form the basic nougat mixture. Furtheringredients such as flavoring agents, additional carbohydrate bulkingagent, coloring agents, preservatives, medicaments, mixtures thereof andthe like may be added thereafter also under agitation. A generaldiscussion of the composition and preparation of nougat confections maybe found in B. W. Minifie, Chocolate, Cocoa and Confectionery: Scienceand Technology, 2nd edition, AVI Publishing Co., Inc., Westport, Conn.(1980), at pages 424-425, which disclosure is incorporated herein byreference.

The procedure for preparing the soft confectionery involves knownprocedures. In general, the frappe component is prepared first andthereafter the syrup component is slowly added under agitation at atemperature of at least about 65° C., and preferably at least about 100°C. The mixture of components is continued to be mixed to form a uniformmixture, after which the mixture is cooled to a temperature below 80°C., at which point, the flavoring agent may be added. The mixture isfurther mixed for an additional period until it is ready to be removedand formed into suitable confectionery shapes.

The ingestible therapeutic wound healing compositions may also be in theform of a pharmaceutical suspension. Pharmaceutical suspensions of thisinvention may be prepared by conventional methods long established inthe art of pharmaceutical compounding. Suspensions may contain adjunctmaterials employed in formulating the suspensions of the art. Thesuspensions of the present invention can comprise:

(a) preservatives such as butylated hydroxyanisole (BHA), butylatedhydroxytoluene (BHT), benzoic acid, ascorbic acid, methyl paraben,propyl paraben, tocopherols, and the like, and mixtures thereof.Preservatives are generally present in amounts up to about 1%, andpreferably from about 0.05% to about 0.5%, by weight of the suspension;

(b) buffers such as citric acid-sodium citrate, phosphoric acid-sodiumphosphate, and acetic acid-sodium acetate in amounts up to about 1%, andpreferably from about 0.05% to about 0.5%, by weight of the suspension;

(c) suspending agents or thickeners such as cellulosics likemethylcellulose, carrageenans like alginic acid and its derivatives,xanthan gums, gelatin, acacias, and microcrystalline cellulose inamounts up to about 20%, and preferably from about 1% to about 15%, byweight of the suspension;

(d) antifoaming agents such as dimethyl polysiloxane in amounts up toabout 0.2%, and preferably from about 0.01% to about 0.1%, by weight ofthe suspension;

(e) sweetening agents such as those sweeteners well known in the art,including both natural and artificial sweeteners. Sweetening agents suchas monosaccharides, disaccharides and polysaccharides such as xylose,ribose, glucose (dextrose), mannose, galactose, fructose (levulose),sucrose (sugar), maltose, invert sugar (a mixture of fructose andglucose derived from sucrose), partially hydrolyzed starch, corn syrupsolids, dihydrochalcones, monellin, steviosides, glycyrrhizin, and sugaralcohols such as sorbitol, mannitol, maltitol, hydrogenated starchhydrolysates and mixtures thereof may be utilized in amounts up to about60%, and preferably from about 20% to about 50%, by weight of thesuspension. Water-soluble artificial sweeteners such as solublesaccharin salts, i.e., sodium or calcium saccharin salts, cyclamatesalts, the sodium, ammonium or calcium salt of3,4-dihydro-6-methyl-1,2,3-oxathiazine-4-one-2,2-dioxide, the potassiumsalt of 3,4-dihydro-6-methyl-1,2,3-oxathiazine-4-one-2,2-dioxide(Acesulfame-K), the free acid form of saccharin, and the like may beutilized in amounts from about 0.001% to about 5%, by weight of thesuspension;

(f) flavoring agents such as those flavors well known to the skilledartisan, such as natural and artificial flavors and mints, such aspeppermint, menthol, citrus flavors such as orange and lemon, artificialvanilla, cinnamon, various fruit flavors, both individual and mixed andthe like may be utilized in amounts from about 0.5% to about 5%, byweight of the suspension;

(g) coloring agents such as pigments which may be incorporated inamounts up to about 6%, by weight of the suspension. A preferredpigment, titanium dioxide, may be incorporated in amounts up to about2%, and preferably less than about 1%, by weight of the suspension. Thecoloring agents may also include natural food colors and dyes suitablefor food, drag and cosmetic applications. These colorants are known asF.D.& C. dyes and lakes. The materials acceptable for the foregoing usesare preferably water-soluble. Such dyes are generally present in amountsup to about 0.25%, and preferably from about 0.05% to about 0.2%, byweight of the suspension;

(h) decolorizing agents such as sodium metabisulfite, ascorbic acid andthe like may be incorporated into the suspension to prevent colorchanges due to aging. In general, decolorizing agents may be used inamounts up to about 0.25%, and preferably from about 0.05% to about0.2%, by weight of the suspension; and

(i) solubilizers such as alcohol, propylene glycol, polyethylene glycol,and the like may be used to solubilize the flavoring agents. In general,solubilizing agents may be used in amounts up to about 10%, andpreferably from about 2% to about 5%, by weight of the suspension.

The pharmaceutical suspensions of the present invention may be preparedas follows:

(A) admix the thickener with water heated from about 40° C. to about 95°C., preferably from about 40° C. to about 70° C., to form a dispersionif the thickener is not water soluble or a solution if the thickener iswater soluble;

(B) admix the sweetening agent with water to form a solution;

(C) admix the therapeutic wound healing composition with thethickener-water admixture to form a uniform thickener-therapeutic woundhealing composition;

(D) combine the sweetener solution with the thickener-therapeutic woundhealing composition and mix until uniform; and

(E) admix the optional adjunct materials such as coloring agents,flavoring agents, decolorants, solubilizers, antifoaming agents, buffersand additional water with the mixture of step (D) to form thesuspension.

The ingestible therapeutic wound healing compositions of this inventionmay also be in chewable form. To achieve acceptable stability andquality as well as good taste and mouth feel in a chewable formulationseveral considerations are important. These considerations include theamount of active substance per tablet, the flavoring agent employed, thedegree of compressibility of the tablet and the organoleptie propertiesof the composition.

Chewable therapeutic candy is prepared by procedures similar to thoseused to make soft confectionery. In a typical procedure, a boiledsugar-corn syrup blend is formed to which is added a frappe mixture. Theboiled sugar-corn syrup blend may be prepared from sugar and corn syrupblended in parts by weight ratio of about 90:10 to about 10:90. Thesugar-corn syrup blend is heated to temperatures above about 120° C. toremove water and to form a molten mass. The frappe is generally preparedfrom gelatin, egg albumin, milk proteins such as casein, and vegetableproteins such as soy protein, and the like, which is added to a gelatinsolution and rapidly mixed at ambient temperature to form an aeratedsponge like mass. The frappe is then added to the molten candy mass andmixed until homogeneous at temperatures between about 65° C. and about120° C.

The ingestible therapeutic wound healing composition of the instantinvention can then be added to the homogeneous mixture as thetemperature is lowered to about 65° C.-95° C. whereupon additionalingredients can then be added such as flavoring agents and coloringagents. The formulation is further cooled and formed into pieces ofdesired dimensions.

A general discussion of the lozenge and chewable tablet forms ofconfectionery may be found in H. A. Lieberman and L. Lathman,Pharmaceutical Dosage Forms: Tablets Volume 1, Marcel Dekker, Inc., NewYork, N.Y. at pages 289 to 466, which disclosure is incorporated hereinby reference.

In accordance with this invention, therapeutically effective amounts ofthe therapeutic wound healing compositions of the present invention maybe admixed into the hard and soft confectionery products. These amountsare readily determined by those skilled in the art without the need forundue experimentation. In a preferred embodiment, the ingestibletherapeutic wound healing composition will comprise the therapeuticwound healing composition in an amount from about 0.1% to about 10% andan ingestible vehicle, that is a pharmaceutically acceptable carrier, ina quantity sufficient to bring the total amount of composition to 100%,by weight the ingestible therapeutic wound healing composition. In amore preferred embodiment, the ingestible composition will comprise thetherapeutic wound healing composition in an amount from about 0.1% toabout 5%, and in a most preferred embodiment, the ingestible compositionwill comprise the therapeutic wound healing composition in an amountfrom about 0.1% to about 2%, and an ingestible vehicle in a quantitysufficient to bring the total amount of composition to 100%, by weightthe ingestible therapeutic wound healing composition.

The present invention extends to methods of making the ingestibletherapeutic wound healing compositions. In such methods, an ingestibletherapeutic wound healing composition is prepared by adrnixing atherapeutically effective amount of the therapeutic wound healingcomposition with a pharmaceutically-acceptable carrier. The apparatususeful in accordance with the present invention comprises mixing andheating apparatus well known in the confectionery arts, and thereforethe selection of the specific apparatus will be apparent to the artisan.The final ingestible therapeutic wound healing compositions are readilyprepared using methods generally known in the confectionery arts.

The therapeutic wound healing compositions may also be incorporated intochewing gums. In this form of the invention, the chewing gum compositioncontains a gum base, a bulking agent, the inventive therapeutic woundhealing composition, and various additives.

The gum base employed will vary greatly depending upon various factorssuch as the type of base desired, the consistency of gum desired and theother components used in the composition to make the final chewing gumproduct. The gum base may be any water-insoluble gum base known in theart, and includes those gum bases utilized for chewing gums and bubblegums. Illustrative examples of suitable polymers in gum bases includeboth natural and synthetic elastomers and rubbers. For example, thosepolymers which are suitable as gum bases include, without limitation,substances of vegetable origin such as chicle, crown gum, nispero,rosadinha, jelutong, perillo, niger gutta, tunu, balata, gutta-percha,lechi-capsi, sorva, gutta kay, mixtures thereof and the like. Syntheticelastomers such as butadiene-styrene copolymers, polyisobutylene,isobutylene-isoprene copolymers, polyethylene, mixtures thereof and thelike are particularly useful.

The gum base may include a non-toxic vinyl polymer, such as polyvinylacetate and its partial hydrolysate, polyvinyl alcohol, and mixturesthereof. When utilized, the molecular weight of the vinyl polymer mayrange from about 2,000 up to and including about 94,000.

The amount of gum base employed will vary greatly depending upon variousfactors such as the type of base used, the consistency of the gumdesired and the other components used in the composition to make thefinal chewing gum product. In general, the gum base will be present inamounts from about 5% to about 94%, by weight of the final chewing gumcomposition, and preferably in amounts from about 15% to about 45%, andmore preferably in amounts from about 15% to about 35%, and mostpreferably in amounts from about 20% to about 30%, by weight of thefinal chewing gum composition.

The gum base composition may contain conventional elastomer solvents toaid in softening the elastomer base component. Such elastomer solventsmay comprise terpinene resins such as polymers of Alpha-pinene orβ-pinene, methyl, glycerol or pentaerythritol esters of rosins ormodified rosins and gums, such as hydrogenated, dimerized or polymerizedrosins or mixtures thereof. Examples of elastomer solvents suitable foruse herein include the pentaerythritol ester of partially hydrogenatedwood or gum rosin, the pentaerythritol ester of wood or gum rosin, theglycerol ester of wood rosin, the glycerol ester of partially dimerizedwood or gum rosin, the glycerol ester of polymerized wood or gum rosin,the glycerol ester of tail oil rosin, the glycerol ester of wood or gumrosin and the partially hydrogenated wood or gum rosin and the partiallyhydrogenated methyl ester of wood or rosin, mixtures thereof, and thelike. The elastomer solvent may be employed in amounts from about 5% toabout 75%, by weight of the gum base, and preferably from about 45% toabout 70%, by weight of the gum base.

A variety of traditional ingredients may be included in the gum base ineffective amounts such as plasticizers or softeners such as lanolin,palmitic acid, oleic acid, stearic acid, sodium stearate, potassiumstearate, glyceryl triacetate, glyceryl lecithin, glyceryl monostearate,propylene glycol monostearate, acetylated monoglyceride, glycerine,mixtures thereof, and the like may also be incorporated into the gumbase to obtain a variety of desirable textures and consistencyproperties. Waxes, for example, natural and synthetic waxes,hydrogenated vegetable oils, petroleum waxes such as polyurethane waxes,polyethylene waxes, paraffin waxes, microcrystalline waxes, fatty waxes,sorbitan monostearate, tallow, propylene glycol, mixtures thereof, andthe like may also be incorporated into the gum base to obtain a varietyof deskable textures and consistency properties. These traditionaladditional materials are generally employed in amounts up to about 30%,by weight of the gum base, and preferably in amounts from about 3% toabout 20%, by weight of the gum base.

The gum base may include effective amounts of mineral adjuvants such ascalcium carbonate, magnesium carbonate, alumina, aluminum hydroxide,aluminum silicate, talc, tricalcium phosphate, dicalcium phosphate andthe like as well as mixtures thereof. These mineral adjuvants may serveas fillers and textural agents. These fillers or adjuvants may be usedin the gum base in various amounts. Preferably the amount of filler whenused will be present in an amount up to about 60%, by weight of thechewing gum base.

The chewing gum base may additionally include the conventional additivesof coloring agents, antioxidants, preservatives and the like. Forexample, titanium dioxide and other dyes suitable for food, drug andcosmetic applications, known as F.D. & C. dyes, may be utilized. Anantioxidant such as butylated hydroxytoluene (BHT), butylatedhydroxyanisole (BHA), propyl gallate, and mixtures thereof, may also beincluded. Other conventional chewing gum additives known to one havingordinary skill in the chewing gum art may also be used in the chewinggum base.

The gum composition may include effective amounts of conventionaladditives selected from the group consisting of sweetening agents(sweeteners), plasticizers, softeners, emulsifiers, waxes, fillers,bulking agents, mineral adjuvants, flavoring agents (flavors,flavorings), coloring agents (colorants, colorings), antioxidants,acidulants, thickeners, mixtures thereof and the like. Some of theseadditives may serve more than one purpose. For example, in sugarless gumcompositions, the sweetener, e.g., sorbitol or other sugar alcohol ormixtures thereof, may also function as a bulking agent. Similarly, insugar containing gum compositions, the sugar sweetener can also functionas a bulking agent.

The plasticizers, softeners, mineral adjuvants, colorants, waxes andantioxidants discussed above as being suitable for use in the gum basemay also be used in the gum composition. Examples of other conventionaladditives which may be used include emulsifiers, such as lecithin andglyceryl monostearate, thickeners, used alone or in combination withother softeners, such as methyl cellulose, alginates, carrageenan,xanthan gum, gelatin, carob, tragacanth, locust bean, and carboxy methylcellulose, acidulants such as malic acid, adipic acid, citric acid,tamtic acid, fumaric acid, and mixtures thereof, and fillers, such asthose discussed above under the category of mineral adjuvants. Thefillers when used may be utilized in an amount up to about 60%, byweight of the gum composition.

Bulking agents (carriers, extenders) suitable for use in chewing gumsinclude sweetening agents selected from the group consisting ofmonosaccharides, disaccharides, poly-saccharides, sugar alcohols, andmixtures thereof; polydextrose; maltodextrins; minerals, such as calciumcarbonate, talc, titanium dioxide, dicalcium phosphate, and the like.Bulking agents may be used in amounts up to about 90%, by weight of thefinal gum composition, with amounts from about 40% to about 70%, byweight of the gum composition being preferred, with from about 50% toabout 65%, by weight, being more preferred and from about 55% to about60%, by weight of the chewing gum composition, being most preferred.

The sweetening agent used may be selected from a wide range of materialsincluding water-soluble sweeteners, water-soluble artificial sweeteners,water-soluble sweeteners derived from naturally occurring water-solublesweeteners, dipeptide based sweeteners, and protein based sweeteners,including mixtures thereof. Without being limited to particularsweeteners, representative categories and examples include:

(a) water-soluble sweetening agents such as monosaccharides,disaccharides and polysaccharides such as xylose, ribulose, glucose(dextrose), mannose, galactose, fructose (levulose), sucrose (sugar),maltose, invert sugar (a mixture of fructose and glucose derived fromsucrose), partially hydrolyzed starch, corn syrup solids,dihydrochalcones, monellin, steviosides, glycyrrhizin, and sugaralcohols such as sorbitol, mannitol, maltitol, hydrogenareal starchhydrolysates and mixtures thereof;

(b) water-soluble artificial sweeteners such as soluble saccharin salts,i.e., sodium or calcium saccharin salts, cyclamate salts, the sodium,ammonim or calcium salt of3,4-dihydro-6-methyl-1,2,3-oxathiazine-4-one-2,2-dioxide, the potassiumsalt of 3,4-dihydro-6-methyl-1,2,3-oxathiazine-4-one-2,2-dioxide(Acesulfame-K), the free acid form of saccharin, and the like;

(c) dipeptide based sweeteners, such as L-aspartic acid derivedsweeteners, such as L-aspartyl-L-phenylalanine methyl ester (Aspartame)and materials described in U.S. Pat. No. 3,492,131,L-Alpha-aspartyl-N-(2,2,4,4-tetramethyl-3-thietanyl)-D-alanin-amidehydrate (Alitame), methyl esters of L-aspartyl- L-phenylglycerineandL-aspartyl-L-2,5-dihydrophenyl-glycine,L-aspartyl-2,5-dihydro-L-phenylalanine;L-aspartyl-L-(1-cyclohexen)-alanine, and the like;

(d) water-soluble sweeteners derived from naturally occurringwater-soluble sweeteners, such as chlorinated derivatives of ordinarysugar (sucrose), known, for example, under the product designation ofSucralose; and

(e) protein based sweeteners such as thaumaoccous danielli (Thaumatin Iand II).

In general, an effective amount of sweetener is utilized to provide thelevel of bulk and/or sweetness desired, and this amount will vary withthe sweetener selected. This amount of sweetener will normally bepresent in amounts from about 0.0025% to about 90%, by weight of the gumcomposition, depending upon the sweetener used. The exact range ofamounts for each type of sweetener is well known in the art and is notthe subject of the present invention. The amount of sweetener ordinarilynecessary to achieve the desired level of sweemess is independent fromthe flavor level achieved from flavor oils.

Preferred sugar base&sweeteners are sugar (sucrose), corn syrup andmixtures thereof. Preferred sugarless sweeteners are the sugar alcohols,artificial sweeteners, dipeptide based sweeteners and mixtures thereof.Preferably, sugar alcohols are used in the sugarless compositionsbecause these sweeteners can be used in amounts which are sufficient toprovide bulk as well as the desired level of sweetness. Preferred sugaralcohols are selected from the group consisting of sorbitol, xylitol,maltitol, mannitol, and mixtures thereof. More preferably, sorbitol or amixture of sorbitol and mannitol is utilized. The gamma form of sorbitolis preferred. An artificial sweetener or dipeptide based sweetener ispreferably added to the gum compositions which contain sugar alcohols.

The coloring agents useful in the gum compositions are used in amountseffective to produce the desired color. These coloring agents includepigments which may be incorporated in amounts up to about 6% by weightof the gum composition. A preferred pigment, titanium dioxide, may beincorporated in amounts up to about 2%, and preferably less than about1% by weight of the composition. The colorants may also include naturalfood colors and dyes suitable for food, drug and cosmetic applications.These colorants are known as F.D.& C. dyes and lakes. The materialsacceptable for the foregoing uses are preferably water-soluble.Illustrative nonlimiting examples include the indigoid dye known asF.D.& C. Blue No.2, which is the disodium salt of5,5-indigotindisulfonic acid. Similarly, the dye known as F.D.& C. GreenNo. 1 comprises a triphenylmethane dye and is the monosodium salt of4-[4-(N-ethyl-p-sulfoninmbenzylamino)diphenylmethylene]-[1-(N-ethyl-N-p-sulfoniumbenzyl)-delta-2,5-cyclohexadieneimine].A full recitation of all F.D.& C. colorants and their correspondingchemical structures may be found in the Kirk-Othmer Encyclopedia ofChemical Technology, 3rd Edition, in volume 5 at pages 857-884, whichtext is incorporated herein by reference.

Suitable oils and fats usable in gum compositions include partiallyhydrogenated vegetable or animal fats, such as coconut oil, palm kerneloil, beef tallow, lard, and the like. These ingredients when used aregenerally present in amounts up to about 7%, by weight, and preferablyup to about 3.5%, by weight of the gum composition.

In accordance with this invention, therapeutically effective amounts ofthe therapeutic wound healing compositions of the present invention maybe admixed into a chewing gum. These amounts are readily determined bythose skilled in the art without the need for undue experimentation. Ina preferred embodiment, the final chewing gum composition will comprisethe therapeutic wound healing composition in an amount from about 0.1%to about 10% and a chewing gum composition in a quantity sufficient tobring the total amount of composition to 100%, by weight of the chewinggum composition. In a more preferred embodiment, the final chewing gumcomposition will comprise the therapeutic wound healing composition inan amount from about 0.1% to about 5%, and in a most preferredembodiment, the final chewing gum composition will comprise thetherapeutic wound healing composition in an amount from about 0.1% toabout 2%, and a chewing gum composition in a quantity sufficient tobring the total amount of composition to 100%, by weight of the chewinggum composition.

The present invention extends to methods of making the therapeuticchewing gum compositions. The therapeutic wound healing compositions maybe incorporated into an otherwise conventional chewing gum compositionusing standard techniques and equipment known to those skilled in theart. The apparatus useful in accordance with the present inventioncomprises mixing and heating apparatus well known in the chewing gummanufacturing arts, and therefore the selection of the specificapparatus will be apparent to the artisan.

For example, a gum base is heated to a temperature sufficiently highenough to soften the base without adversely eftcoting the physical andchemical make up of the base. The optimum temperatures utilized may varydepending upon the composition of the gum base used, but suchtemperatures are readily determined by those skilled in the art withoutundue experimentation.

The gum base is conventionally melted at temperatures that range fromabout 60° C. to about 120° C. for a period of time sufficient to renderthe base molten. For example, the gum base may be heated under theseconditions for a period of about thirty minutes just prior to beingadmixed incrementally with the remaining ingredients of the base such asthe plasticizer, fillers, the bulking agent and/or sweeteners, thesoftener and coloring agents to plasticize the blend as well as tomodulate the hardness, viscoelasticity and formability of the base. Thechewing gum base is then blended with the therapeutic wound healingcomposition of the present invention which may have been previouslyblended with other traditional ingredients. Mixing is continued until auniform mixture of gum composition is obtained. Thereafter the gumcomposition mixture may be formed into desirable chewing gum shapes.

In a specific embodiment, the invention is directed to a therapeuticpharmaceutical composition for preventing and reducing injury tomammalian cells, and increasing the resuscitation rate of injuredmammalian cells, which comprises:

(A) a therapeutically effective amount of a therapeutic wound healingcomposition of Embodiment One (I) selected from the group consisting of:

(I.A)

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.B)

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.C)

(a) an antioxidant; and

(b) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.D)

(a) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier may be selected from the groupconsisting of pharmaceutical appliances, topical vehicles, andingestible vehicle.

In another specific embodiment, the invention is directed to a methodfor preparing a therapeutic pharmaceutical composition for preventingand reducing injury to mammalian cells, and increasing the resuscitationrate of injured mammalian cells, which comprises the steps of:

(A) providing a therapeutically effective amount of a therapeutic woundhealing composition of Embodiment One (I.A-D) selected from the groupconsisting of:

(I.A)

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.B)

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.C)

(a) an antioxidant; and

(b) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.D)

(a) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) providing a pharmaceutically acceptable carrier; and

(C) admixing the therapeutic wound healing composition from step (A) andthe pharmaceutically acceptable carrier from step (B) to form atherapeutic pharmaceutical composition.

Throughout this application, various publications have been referenced.The disclosures in these publications are incorporated herein byreference in order to more fully describe the state of the art.

The present invention is further illustrated by the following exampleswhich are not intended to limit the effective scope of the claims. Allparts and percentages in the examples and throughout the specificationand claims are by weight of the final composition unless otherwisespecified.

E. Examples Of The Therapeutic Wound Healing Compositions Of EmbodimentOne (I.A-D) Study 1

This study demonstrates a comparison of the viability of U937 monocyticcells after exposure of the cells to various antioxidants andcombinations of antioxidants. This study also demonstrate a comparisonof the levels of hydrogen peroxide produced by U937 monocytic cells andmammalian epidermal keratinocytes after exposure of the cells to variousantioxidants and combinations of antioxidants. The results of this studyare illustrated in FIGS. 1-4 and examples 1-26 below.

Mammalian epidermal keratinocytes and monocytes were employed to examinethe ability of various antioxidants to reduce levels of hydrogenperoxide in these cells. Hydrogen peroxide was measured after the cellswere exposed to ultraviolet light in the wavelength range from 290 to320 nm (UV-B) or to the inflammatory compound12-0-tetradecanoyl-phorbol-13-acetate (TPA). Sodium pyruvate was testedat various concentrations to determine the effect of concentrations ofthis antioxidant on the hydrogen peroxide production by epidermal cellsand monocytes. Magnesium pyruvate, calcium pyruvate, zinc pyruvate, andcombinations of sodium pyruvate with ascorbic acid, lactic acid, andVitamin E were then tested to determine the effect of these salts andcombinations of antioxidants on the hydrogen peroxide production byepidermal cells and monocytes.

Mammalian epidermal kemtinocytes were isolated by trypsinization ofepithelial sheets and grown in modified basal MCDB 153 mediumsupplemented with epidermal growth factor, bovine pituitary extract, andhydrocortisone. Cells were maintained in a humidified incubator with 5%carbon dioxide at 37° C. Keratinocytes were seeded in 60 mm culturedishes at a cell density of 3×10⁵ cells per dish and the cultures wereexposed to 1 M.E.D. dose of ultraviolet-B light (100 mJ/cm²) or treatedwith 100 ng/ml of TPA.

U937 monocytic cells are a cultured cell line grown in RPMI media with10% fetal calf serum. Cells were maintained in a 60 mm culture dish at5% carbon dioxide at 37° C. at a seeding density not exceeding 1×10⁶cells per dish.

Sodium pyruvate, lactic acid, ascorbic acid, and Vitamin E weredissolved in distilled water, with sufficient surfactant. Theconcentrations of the sodium pyruvate solutions prepared were 1 mM, 10mM, 50 mM, 100 mM, and 200 mM. The concentrations of the lactic acidsolutions prepared were 1.0%, 0.1%, and 0.05%. The concentrations of theascorbic acid solutions prepared were 1.0%, 0.1%, 0.05%, and 0.025%. Theconcentrations of the Vitamin E solutions prepared were 1 U, 10 U, 50 U,and 100 U. The test solutions were adjusted to a pH value of 7.4 with1.0N sodium hydroxide solution and then sterile filtered. Theappropriate concentration of test solution or combination of testsolutions was added to the cells immediately prior to exposure of thecells to ultraviolet light-B or TPA [100 ng/ml]. Stock solutions wereprepared so that the vehicle did not constitute more than 1% of thetotal volume of the culture media.

Intracellular hydrogen peroxide production by mammalian epidermalkeratinocytes and U937 monoceytes was measured using dichlorofluoresceindiacetate (DCFH-DA, Molecular Probes, Eugene, Oreg.). DCFH-DA is anon-polar nonfluorescent compound that readily diffuses into cells whereit is hydrolyzed to the polar non-fluorescent derivative DCFH which thenbecomes trapped within the cells. In the presence of intracellularhydrogen peroxide, DCFH is oxidized to the highly fluorescent compoundDCF. Hence, cellular fluorescence intensity is directly proportional tothe level of intracellular hydrogen peroxide produced. Cellularfluorescence intensity can be monitored by fluorimetry and by flowcytometry.

Mammalian epidermal keratinocytes and U937 cultured monocytes (1×10⁶ perdish) were incubated at 37° C. with 5 uM of DCFH-DA. Production ofhydrogen peroxide was measured using a Coulter Profile analytical flowcytometer. Linear and log intensity of green fluorescence data wascollected. For each analysis, a quantity of 10,000 to 20,000 events wasaccumulated. Optical alignment for the instrument was performed daily.Coefficients of variation for forward angle light scatter and integratedgreen fluorescence were generally less than two. Each analysis wasrepeated three times and the quantitation of fluorescence was expressedin terms of femtomoles (fmol, 10⁻¹⁵ moles) of DCF oxidized per cell,which is a direct measure of the intracellular hydrogen peroxideproduced. Alternatively, in the saturated and unsaturated fatty acidexamples in examples 27-52, fluorimetry was used to assess the DCFoxidation per cell.

The viability of the U937 monocytic cells after exposure of the cells tovarious antioxidants for 24 hours was measured. The viability of thecells was determined by exposing the cells to the dye propidium iodide.Permeable cell membranes which absorbed the dye were not consideredviable. The viability of the cells was represented as the percentage ofcells that excluded propidium iodide. FIG. 1 depicts in bar graph formatthe viability of U937 monocytic cells after exposure of the cells to noantioxidant (Example 1, control), to sodium pyruvate (Example 2), toascorbic acid (Example 3), to lactic acid (Example 4), and to Vitamin E(Example 5). FIG. 2 depicts in bar graph format the viability of U937monocytic cells after exposure of the cells to various combinations ofantioxidants. Specifically, the viability of U937 monocytic cells wasmeasured after exposure to no antioxidant (Example 6, control), toascorbic acid and lactic acid (Example 7), to ascorbic acid and VitaminE (Example 8), to sodium pyruvate and ascorbic acid (Example 9), tosodium pyruvate and lactic acid (Example 10), to sodium pyruvate andVitamin E (Example 11), to lactic acid and Vitamin E (Example 12), andto sodium pyruvate, ascorbic acid, and lactic acid (Example 13).

FIG. 1 shows that ascorbic acid is cytotoxic to monocytes atconcentrations as low as 0.25%. FIG. 2 shows that the cytotoxicity ofascorbic acid was reversed by the addition of 10 mM of sodium pyruvate.FIGS. 1 and 2 show that the viability rate of 15% to 20% of the cellswhen treated with ascorbic acid was increased to 95% to 98% uponaddition of sodium pyruvate. Lactic acid and Vitamin E did not reversethe cytotoxicity of ascorbic acid.

Sodium pyruvate was then tested at various concentrations to determinethe effect of concentrations of this antioxidant on the hydrogenperoxide production by epidermal cells and monocytes. Mammalianepidermal keratinocytes and monocytes were exposed to (a) 1 M.E.D. doseof ultraviolet light-B and (b) 100 ng/ml of12-O-tetradecanoylphorbol-13-acetate (TPA) in the presence of sodiumpyruvate at the following concentrations: 200 mM, 100 mM, 50 mM, 10 mM,1 mM.

The optimum concentration of sodium pyruvate to reduce the hydrogenperoxide production by epidermal cells and monocytes was found to be 10mM. Concentrations of sodium pyruvate of 50 mM and above were cytotoxicto both epidermal keratinocytes and monocytes.

Magnesium pyruvate, calcium pyruvate, zinc pyruvate, ascorbic acid,lactic acid, and Vitamin E, and combinations of sodium pyruvate withascorbic acid, lactic acid, and Vitamin E were then tested to determinethe effect of these salts and combinations of antioxidants on thehydrogen peroxide production by epidermal cells and monocytes. Thefollowing test solutions were prepared.

(a) sodium pyruvate [10 mM];

(b) zinc salt [10 mM];

(c) magnesium salt [10 mM];

(d) calcium salt [10 mM];

(e) sodium pyruvate [10 mM] and ascorbic acid [0.025%];

(f) sodium pyruvate [10 mM] and lactic acid [0.05%];

(g) sodium pyruvate [10 mM], lactic acid, [0.05%], and astorhie acid[0.025%];

(h) lactic acid [1.0%, 0.1%, and 0.05%];

(i) ascorbic acid [1.0%, 0.1%, 0.05%, and 0.025%];

(j) Vitamin E [1 U, 10 U, 50 U, and 100 U]; and

(k) vehicle solvent controls.

There was no significant difference among the zinc, magnesium, andcalcium salts of pyruvic acid on the hydrogen peroxide production byepidermal cells and monocytes. The zinc and calcium salts of pyruvicacid induced differentiation of keratinocytes. For convenience, thesodium salt was used in subsequent tests.

The optimum concentration of lactic acid to reduce the hydrogen peroxideproduction by epidermal cells and monocytes was found to be 0.05%. Theoptimum concentration of ascorbic acid was found to be 0.025%. Thehigher concentrations of both of these compounds were found to becytotoxic to both types of cells. The optimum concentration of Vitamin Ewas found to be 50 U.

FIG. 3 depicts in bar graph format the levels of hydrogen peroxideproduced by U937 monocytic cells after exposure of the cells to noantioxidant (Example 14, control), to sodium pyruvate (Example 15), toascorbic acid (Example 16), to lactic acid (Example 17), and to VitaminE (Example 18). Sodium pyruvate and Vitamin E significantly reduced thehydrogen peroxide production by monocytes.

FIG. 4 depicts in bar graph format the levels of hydrogen peroxideproduced by U937 monocytic cells after exposure of the cells to variouscombinations of antioxidants. Specifically, the levels of hydrogenperoxide produced by U937 monocytic cells were measured after exposureto no antioxidant (Example 19, control), to ascorbic acid and lacticacid (Example 20), to ascorbic acid and Vitamin E (Example 21), tosodium pyruvate and ascorbic acid (Example 22), to sodium pyruvate andlactic acid (Example 23), to sodium pyruvate and Vitamin E (Example 24),to lactic acid and Vitamin E (Example 25), and to sodium pyruvate,ascorbic acid, and lactic acid (Example 26). The combination of lacticacid (0.05%) and Vitamin E (50 U) significantly reduced the hydrogenperoxide production by monocytes.

The morphological alterations in epidermal keratinocytes were observedin control cultures and in cultures exposed to ultraviolet-B. Cells inthe layer closest to the dermis are basal keratinocytes. These cellsproliferate and migrate into the spinous and granular layers of theepidermis where the cells begin to differentiate. The differentiationpattern results in cells enucleating and forming cornified envelopes atthe uppermost portion of the epidermis, the statum corneum. Thedifferentiation of keratinocytes is controlled by the levels of calcium,magnesium, and other elements in the medium. Cells in culture systemspromoting differentiation appear as an epidermal sheet formingattachments or tight junctions with each other. Keratinocytes thatbecome nonadherent or float in the media were considered responding to acytotoxic event.

The following morphological alterations in the mammalian epidermalkeratinocytes were observed for the following control cultures:

10 mM Sodium Pyruvate: Tight junctions of cells were formed and theproliferation rate of the cells was higher than the rate of the controlcells.

0.025% Ascorbic Acid: Cells were floating in a cytotoxic response toascorbic acid.

0.025% Ascorbic acid and 10 mM Sodium Pyruvate: Few tight junctions ofcells were observed and cells appeared similar to the cells in thesodium pyruvate culture.

0.05% Lactic Acid: Cells appeared dramatically altered as an epidermalsheet and as flat granular cells.

0.05% Lactic Acid and 10 mM Sodium Pyruvate: Cells formed an epidermalsheet but appeared smaller than the cell in the lacdc acid culture.

50 U Vitamin E: Cells appeared the same as the cells in the controlculture.

50 U Vitamin E and 10 mM Sodium Pyruvate: Cells increased in number andchanged in appearance resembling the cells in the sodium pyruvateculture.

The following morphological alterations in the mammalian epidermalkeratinocytes were observed for the corresponding cultures exposed toultraviolet light-B, 100 mJoules, for 24 hours:

10 mM Sodium Pyruvate: Cells proliferated more rapidly than the cells inthe control culture.

0.025% Ascorbic Acid: Cells were nonadherent and floating in a cytotoxicresponse to ascorbic acid greater than the cytotoxic response of thecorresponding cells without ultraviolet-B light exposure.

0.05% Lactic Acid: Cells formed an epidermal sheet and were moregranular than cells in the control culture without ultraviolet-B lightexposure.

50 U Vitamin E: Cell growth was inhibited but cells appeared similar tocells in the control culture without ultraviolet-B light exposure.

50 U Vitamin E and 10 mM Sodium Pyruvate: Cells appeared similar tocells in the control culture and proliferated to a greater extent thancells in the control cultures without ultraviolet-B light exposure.

Morphological alterations in the U937 monocytic cell line were alsoobserved for control cultures and cultures exposed to ultravioletlight-B, 100 mJoules, for 24 hours. The following compounds andcombination of compounds, at the concentrations set out below,significantly inhibited the levels of hydrogen peroxide produced by U937monocytic cells

    Sodium pyruvate at 10 mM and 50 mM;

    Vitamin E at 50 U and 100 U; and

    Lactic acid at 0.05% and Vitamin E at 50 U.

Examples Of The Therapeutic Wound Healing Compositions Of Embodiment One(I.A-D) Study 2

This study demonstrates a comparison of the levels of hydrogen peroxideproduced by U937 monocytic cells and epidermal keratinocytes afterexposure of the cells to various combinations of antioxidants with andwithout a mixture of saturated and unsaturated fatty acids. The resultsof this study are illustrated in FIGS. 5-7 and examples 27-52 below.

Mammalian epidermal keratinocytes and U937 monocytic cells and the testsolutions of sodium pyruvate, lactic acid, ascorbic acid, and Vitamin Ewere prepared as describe above for Examples 1-26. Intracellularhydrogen peroxide production by the mammalian epidermal keratinocytesand U937 monocytes was also measured as described above.

A mixture of fatty acids derived from chicken fat was prepared foraddition to the cultured cells by mixing 0.1% of the chicken fat withthe culture media. At the temperature of the culture media, 37° C., thechicken fat was miscible. This chicken fat mixture was added to culturesof cells prior to exposure of the cells to ultraviolet-B light or TPAtreatment.

As set out in examples 1-26, mammalian epidermal keratinocytes andmonocytes were exposed to (a) 1 M.E.D. dose of ultraviolet light-B and(b) 100 ng/ml of 12-O-tetradecanoylphorbol-13-acetate in the presence ofvarious antioxidants and combinations of antioxidants with and without amixture of saturated and unsaturated fatty acids [0.1%, 0.5%, and 1.0%chicken fat].

FIG. 5 depicts in bar graph format the levels of hydrogen peroxideproduced by U937 monocytic cells after exposure of the cells to variouscombinations of antioxidants with and without a mixture of saturated andunsaturated fatty acids. Specifically, the levels of hydrogen peroxideproduced by U937 monocytic cells were measured after exposure to lacticacid and Vitamin E without fatty acids (Example 27) and with fatty acids(Example 28), to ascorbic acid and lactic acid without fatty acids(Example 29) and with fatty acids (Example 30), and to ascorbic acid andVitamin E without fatty acids (Example 31) and with fatty acids (Example32). The ability of the combinations of lactic acid and Vitamin E,ascorbic acid and lactic acid, and ascorbic acid and Vitamin E to reducethe hydrogen peroxide production by monocytes was increased in thepresence of fatty acids. The most effective combination to reduce thehydrogen peroxide production of monocytes was lactic acid (0.05%) andVitamin E (50 E) in the presence of a mixture of saturated andunsaturated fatty acids (0.5%).

FIG. 6 depicts in bar graph format the levels of hydrogen peroxideproduced by epidermal keratinocytes after exposure of the cells tovarious antioxidants with and without a mixture of saturated andunsaturated fatty acids. Specifically, the levels of hydrogen peroxideproduced by epidermal keratinocytes were measured after exposure to noantioxidant without fatty acids (Example 33, control) and with fattyacids (Example 34), to sodium pyruvate without fatty acids (Example 35)and with fatty acids (Example 36), to ascorbic acid without fatty acids(Example 37) and with fatty acids (Example 38), to lactic acid withoutfatty acids (Example 39) and with fatty acids (Example 40), and toVitamin E without fatty acids (Example 41) and with fatty acids (Example42). The ability of sodium pyruvate and Vitamin E to reduce the hydrogenperoxide production by epidermal keratinocytes was increased in thepresence of fatty acids. The most effective combinations to reduce thehydrogen peroxide production of epidermal keratinocytes were sodiumpyruvate in combination with a mixture saturated and unsaturated fattyacids and Vitamin E in combination with a mixture of saturated andunsaturated fatty acids.

FIG. 7 depicts in bar graph format the levels of hydrogen peroxideproduced by epidermal keratinocytes after exposure of the cells tovarious combinations of antioxidants with and without a mixture ofsaturated and unsaturated fatty acids. Specifically, the levels ofhydrogen peroxide produced by epidermal keratinocytes were measuredafter exposure to no antioxidant without fatty acids (Example 43,control) and with fatty acids (Example 44), to sodium pyruvate andascorbic acid without fatty acids (Example 45) and with fatty acids(Example 46), to sodium pyruvate and lactic acid without fatty acids(Example 47) and with fatty acids (Example 48), to sodium pyruvate andVitamin E without fatty acids (Example 49) and with fatty acids (Example50), and to ascorbic acid and Vitamin E without fatty acids (Example 51)and with fatty acids (Example 52). The ability of all combinations ofantioxidants to reduce the hydrogen peroxide production by epidermalkemtinocytes was increased in the presence of fatty acids. In order ofpotency, the most effective combinations to reduce the hydrogen peroxideproduction of epidermal keratinocytes were sodium pyruvate and VitaminE, sodium pyruvate and lactic acid, and Vitamin E, each in combinationwith a mixture of saturated and unsaturated fatty acids (0.5%).

Because of the cytotoxicity of cells towards ascorbic acid describedabove, the ascorbic acid combinations without sodium pyruvate were notconsidered significantly different from the control test solution.

Summary Anaylsis of the Data from Studies 1 and 2

Human epidermal keratinocytes were isolated by trypsinization ofepithelial sheets and grown in modified base MCDB 153 mediumsupplemented with epidermal growth factor and bovine pituitary extract.Cells were seeded in culture dishes at a density of 3×10⁵ /dish. Priorto exposure to UV B light (100 mJ/cm²) or treatment with 100 ng/ml TPA,the cultures were treated with the appropriate concentration of woundhealing components. Intracellular production of hydrogen peroxide wasmeasured using DCFH-DA, a nonpolar compound that readily diffuses intocells, hydrolyzed to a nonpolar derivative. In the presence ofintracellular hydrogen peroxide, DCFH is oxidized to a highlyfluorescent compound DCF. Thus, cellular fluorescence intensity isdirectly proportional to levels of hydrogen peroxide produced and can bemonitored by flow cytometry. Hydrogen peroxide is cytotoxic, thereforelower levels of hydrogen peroxide production is desirable for cellularviability.

In all cases, the three component wound healing composition surpassedthe predicted outcomes, clearly demonstrating unpredicted synergy.

    ______________________________________    Results    1                2         3      4    ______________________________________    1 -   Control        250       250   0    2 -   Fatty Acids    250       230   -20          (0.5%)    3 -   Sodium Pyruvate                         250       490  +240          (10 mM)    4 -   Vitamin E      250       400  +150          (50 units)    5 -   Pyruvate &     250       430  +180          Fatty Acids    6 -   Vitamin E &    250       200   -50          Fatty Acids    7 -   Pyruvate &     250       290   +40          Vitamin E    8 -   Pyruvate &     250       120  -130          Vitamin E & Fatty Acids    ______________________________________     Column 1 shows the different treatment groups.     Column 2 shows the production of H.sub.2 O.sub.2 in control cells     (fmol/cell).     Column 3 shows the production of H.sub.2 O.sub.2 after treatment with     wound healing components.     Column 4 shows the difference in production of H.sub.2 O.sub.2 from     control after the treatment.

All comparisons were assessed against the controls, which produced 250H₂ O₂ fmol/cell. The positive numbers represent H₂ O₂ production inexcess of the control and the negative numbers represent H₂ O₂production below the control. These results are set out in FIG. 8.

    ______________________________________    Fatty Acids (-20) & Vitamin E (+150) & Pyruvate (+240)    +370 Is The Predicted Three Component Effect    -130 Is The Wound healing composition Actual Effect    500 Is The Difference Between Predicted Effect minus Actual effect    (Synergy)    Combination of Paired and Single Ingredients    Pyruvate & Fatty Acids (+180) & vitamin E (+150)    +330 Is The Predicted Three Component Effect    -130 Is The Wound healing composition Actual Effect    460 Is The Difference between Predicted Effect minus Actual Effect    (Synergy)    Vitamin E & Fatty Acids (-50) & Pyruvate (+240)    +190 Is The Predicted Three Component Effect    -130 Is The Wound healing composition Actual Effect    320 Is The Difference between Predicted Effect minus Actual Effect    (Synergy)    Pyruvate & Vitamin E (+40) & Fatty Acids (-20)    +20 Is The Predicted Three Component Effect    -130 Is The Wound healing composition Actual Effect    150 Is The Difference between Predicted Effect minus Actual Effect    (Synergy)    ______________________________________

In all cases, the three component wound healing composition surpassedthe predicted outcomes clearly demonstrating unpredicted synergy.

Examples of the Therapeutic Wound Healing Compositions of Embodiment One(I.A-D) Study 3

This study demonstrates a comparison of the wound healing abilities ofthe therapeutic wound healing compositions of the present inventionversus conventional wound healing compositions. The results of thisstudy are illustrated in examples A-D.

The wound healing compositions of Examples A-D were prepared having thecompositions set out in Table A.

    ______________________________________    Examples                A    Ingredient  Prep-H ™                         B         C     D    ______________________________________    sodium pyruvate                --        2%       --    --    vitamin E   --        1%       --    --    chicken fat --        2%       --    --    LYCD        2000 U*  2400 U    2400 U                                         --    shark liver oil                3%*       3%          3% --    petrolatum  in       64%        66.5%                                         68%    mineral oil amounts  22.53%    25.03%                                         26.8%    paraffin    totaling  5%          5%  5%    emulsifier  100%     0.2%       0.2% 0.2%    ______________________________________     *These components are present in Preparation H

Wound healing composition A was commercially available Preparation H™.Wound healing composition B was a petrolatum base formulation containinglive yeast cell derivative, shark oil, and a mixture of sodium pyruvate,vitamin E, and chicken fat. Wound healing composition C was a petrolatumbase formulation containing live yeast cell derivative and shark oil.Wound healing composition D was a petrolatum base formulation only.

Wound healing studies were carried out using hairless mice (SKR-1,Charles River) 6-8 weeks in age. One group of mice were untreated as acontrol group and were referred to as Example E. In each group therewere 6 mice for evaluation at either day 3 or day 7 for a total numberof 60 animals in the study. The mice were anesthetized with ether and amidline 3 cm full thickness longitudinal incision was made with a number10 scalpel blade. Incisions were closed using steel clips at 1 cmintervals. Formulations A-D set out above were applied in a randomizedblinded study to the wounds on day 0 at 2 hours following wounding andreapplied at 24 hour intervals during the 7 days of the study. Thewounds were examined daily and scored on a basis of 0-5 for closure oneach day of the study, with a score of 5 representing the wound besthealed.

The animals were sacrificed on day 3 and day 7 using cervicaldislocation. The dorsal skin including the incision was dissectedwithout the subcutaneous tissue. The skin was placed in neutral bufferedformalin and subsequently sectioned and stained with hematoxylin andeosin. The wounds were examined microscopically and representativetissue sections were photographed.

On each day of the experiment, the score and rank order of theformulations for closure of wounds and speed of healing were as follows:

    B (5)>>D (4)>>C (2)>/=E, Control (2)>A (1)

Photographs of the wounded mice on day 4 are set out in FIGS. 9A-9D and10.

FIGS. 9A-9D and 10 show that Formulation B, which was a petrolatum baseformulation containing live yeast cell derivative, shark oil, and amixture of sodium pyruvate, vitamin E, and chicken fat, was asignificantly better wound healing agent than the other formulations.These results are supported by the subjective grading of the woundclosures and the speed of healing on each day (1-7) of the experiment aswell as on the objective histological examination of tissue sections tomeasure the extent of inflammatory cell infiltrate within the wound andthe extent of epithelialization at the wound edges. The final result wasthat less scar tissue was present at day 7 on the mice treated withFormulation B.

Formulation D, which was a white petrolatum formulation only, was judgedto be significantly more effective to promote healing than eitherFormulation C, which was a petrolatum base formulation containing sharkliver oil and live yeast cell derivative, or Formulation A, which wasPreparation H™. The superior ability of Formulation D over Formulation Cto improve healing may result from a delay in the healing process causedwhen the live yeast cell derivative is depleted and the cells shift toan alternative nutrient source. The presence of the mixture of sodiumpyruvate, vitamin E, and chicken fat in Formulation B apparently offsetsthe depletion of the live yeast cell derivative.

Formulation C, which was a petrolatum base formulation containing liveyeast cell derivative and shark oil, was judged comparable to thecontrol (untreated wound) in speed of wound closure and extent ofhealing. Formulation A, which was Preparation H™, appeared to be theleast effective healing formulation by both subjective grading of woundhealing and by objective examination of tissue sections. The superiorability of Formulation D and Formulation C over Formulation A to improvehealing may be due to their ability to act as an occlusive wounddressing that prevents transepidermal water loss and thus promoteshealing and wound closure. The poor ability of Formulation A to improvehealing may be due to the potential cytotoxicity of phenylmercuricnitrate present in Preparation H™ as a preservative.

These results show that the wound healing compositions of the presentinvention which comprise a mixture of sodium pyruvate, vitamin E, andchicken fat increase the proliferation and resuscitation rate ofmammalian cells. The wound healing compositions mediate low levels ofoxygen in the initial stages of healing to suppress oxidative damage andhigher levels of oxygen in the later stages of healing to promotecollagen formation.

II. Augmented Wound Healing Compositions A. Embodiment Two (I.A-D+M)

In Embodiment Two (II), the therapeutic wound healing compositions ofEmbodiment One (I.A-D) are combined with a medicament (M) which isuseful for treating injured mammalian cells to form augmented woundhealing compositions (I.A-D+M) having an enhanced ability to prevent andreduce injury to mammalian cells and further increase the resuscitationrate of injured mammalian cells. The tissue damage associated with manydiseases and conditions such as autoimmune disease and benign andmalignant skin growths is believed to be caused by the production ofcellular produced active oxygen species. Combination of the therapeuticwound healing compositions of the present invention and medicamentsuseful for treating such diseases and conditions may suppress suchreactive oxygen-linked tissue injury.

For example, the therapeutic wound healing compositions may be used intopical augmented wound healing compositions in combination withmedicaments useful for treating wounds such as immunostimulating agents(Betafectin™), antiviral agents, antikeratolytic agents,anti-inflammatory agents, antifungal agents, tretinoin, sunscreenagents, dermatological agents, topical antihistamine agents,antibacterial agents, bioadhesive agents, respiratory burstinginhibitors (lactic acid, adenosine), inhibitors of prostaglandinsynthesis (ibuprofen, aspirin, indomethacin, meclofenomic acid, retinoicacid, padimate O, meclomen, oxybenzone), steroidal anti-inflammatoryagents (corticosteroids including synthetic analogs), antimicrobialagents (neosporin ointment, silvadine), antiseptic agents, anestheticagents (pramoxine hydrochloride, lidocaine, benzocaine), cell nutrientmedia, burn relief medications, sun burn medications, acne preparations,insect bite and sting medications, wound cleansers, wound dressings,scar reducing agents (vitamin E), and the like, and mixtures thereof, tofurther enhance the proliferation and resuscitation rate of mammaliancells. Preferably, the medicament useful for treating wounds is selectedfrom the group consisting of immunostimulating agents, antiviral agents,antikeratolytic agents, anti-inflammatory agents, antifungal agents,tretinoin, sunscreen agents, dermatological agents, topicalantihistamine agents, antibacterial agents, bioadhesive agents,respiratory bursting inhibitors, inhibitors of prostaglandin synthesis,antimicrobial agents, cell nutrient media, scar reducing agents, andmixtures thereof. More preferably, the medicament useful for treatingwounds is selected from the group consisting of immunostimulatingagents, antiviral agents, antikeratolytic agents, anti-inflammatoryagents, antifungal agents, acne treating agents, sunscreen agents,dermatological agents, antihistamine agents, antibacterial agents,bioadhesive agents, and mixtures thereof.

The therapeutic wound healing compositions may also be used iningestible augmented wound healing compositions in combination withmedicaments used to treat injured mammalian cells such as strokemedications; autoimmune disease medications; arthritis medications;ulcer medications; cancer medications (cytotoxic agents); heartmedication to improve regional ventricular function and restore normalheart rate and pressure functions; lung medication to repair injuredtissue; liver medication to suppress lipogenesis of alcoholic origin andprevent hepatic steatosis; kidney medication to suppress urinary calculi(kidney stones); detoxification medication to antagonize heavy metalpoisoning, cyanide poisoning, sodium sulfide poisoning, other types ofpoisoning; and reduce and neutralize the production of oxygen radicalswhich produces injury to tissue, to protect and further enhance theresuscitation rate of the injured mammalian cells.

A cell nutrient medium provides a complete diet of nutrients necessaryfor wound healing. The cell nutrient medium may be derived from animal,plant, and yeast sources. Typical cell nutrient media includes liveyeast cell derivative, Eagles medium, and artificial serum. A preferredcell nutrient medium is live yeast cell derivative. Live yeast cellderivative supplies skin respiratory factor which acts by increasing theoxygen uptake of dermal tissues and facilitates collagen formation. Liveyeast cell derivative generally contains numerous amino acids forcollagen formation, mono- and disaccharides as carbon sources, vitamins,minerals, phosphorous containing compounds, nucleosides, nucleotides,and salts. In general, the amino acids present in live yeast cellderivative include aspartic acid, glutamic acid. histidine, serine,glycine, alanine, arginine, tyrosine, valine, methionine, isoleucine,leucine, phenylalanine, and lysine. The coenzymes present in live yeastcell derivative include vitamin A, vitamin E, vitamin D₃, folic acid,pantothenic acid, niacinamide, vitamin B₁, vitamin B₂, vitamin B₆, andvitamin B₁₂. The cofactor type minerals present in live yeast cellderivative include calcium, copper, iron, magnesium, zinc, andphosphorus. A preferred tissue respiratory factor is Biodynes® TRF,commercially available from Brooks Industries, Inc., South Plainfield,N.J. In general, the cell nutrient medium will be present in thetherapeutic composition in an amount from about 0.01% to about 5%,preferably from about 0.1% to about 1%, and more preferably from about0.2% to about 0.4%, by weight of the therapeutic composition.

Infected wounds are generally treated with an antibiotic agent, anantifungal agent, or an antiviral agent to kill the infecting organism.But these agents do not facilitate healing. Combination of the woundhealing compositions of Embodiment One (I.A-D) with an antibiotic agent,an antifungal agent, or an antiviral agent would provide an augmentedwound healing composition (I.A-D+M) which would kill the infectingorganism and facilitate healing. The wound healing compositions can beused in combination with (a) an antibacterial agent; (b) an antifungalagent for athlete's foot (Clotrimazole), infected toes, jock itch, andvaginal infections; (c) an antiviral agent for cold sores (Acyclovir),genital lesions, and HIV lesions (AZT) (heals and decreases duration andseverity of viral lesions).

Damage from UV light can be avoided through the use of antioxidants.Combination of the wound healing compositions of Embodiment One (I.A-D)with an anti-inflammatory agent would provide an augmented wound healingcomposition (I.A-D+M) which would repair and reduce the erythema causedby the inflammatory responses to overexposure to UV light. The augmentedwound healing compositions can be used for UV protection with SPFagents, UV repair (protects and repairs damaged skin), and antiaging(protects skin from UV damage and aging).

Certain therapeutic drugs are known to have irritating side effects.Combination of the wound healing compositions of Embodiment One (I.A-D)with a drugs that has irritating side effects would provide an augmentedwound healing composition (I.A-D+M) which would have therapeutic value.For example, the wound healing compositions can be used in combinationwith Retin A (reduces irritation of actives) and Vitamin D and analogs(reduces irritation of actives).

Lips are often protected from UV damage with lip balms that containmoisturizing agents and antioxidants. Combination of the wound healingcompositions of Embodiment One (I.A-D) with moisturizing agents wouldprovide an augmented wound healing composition (I.A-D+M) which wouldprovide a healing benefit for dry cracking lips. The augmented woundhealing compositions can be used for lip protection (provides protectionand repair from UV light, cold etc), lip moisturizing (provides amoisture barrier), and mouth sores in combination with an antiseptic(heals sores and kills the germs).

Gingivitis is initiated by supragingival plaque. Uncontrolledsupragingival plaque releases toxins and microbial products that attackthe gingiva and result in inflammation of the gingival tissues.Inflammation in the connective tissues results in pocket formation andmay ultimately result in periodontitis. Oxygen radicals at the site ofinfection can cause tissue damage. Combination of the wound healingcompositions of Embodiment One (I.A-D) with anti-inflammatory agents orantimicrobial agents would provide an augmented wound healingcomposition (I.A-D+M) which may help decrease inflammation and improvehealing of damaged tissues. The augmented wound healing composition canbe used in tooth paste (enhances healing of bleeding gums); mouth washes(helps maintain healthy gums); bioadhesive films (provides long termhealing agents for injured gums); and products to heal cuts fromorthodontic appliances.

Sore throats can be caused by many factors including simple irritationdue to shouting, infections, smoking, or eating the wrong foods.Combination of the wound healing compositions of Embodiment One (I.A-D)with a throat lozenge would provide an augmented wound healingcomposition (I.A-D+M) which would soothe and heal the throat with andwithout antimicrobial agents. The wound healing composition can be usedfor irritated sore throats (soothes and heals inflamed sore throats); incombination with antivirals (soothes and heals inflamed tissues); and incombination with antimicrobials (kills the infection, heals the sorethroat faster).

Combination of the wound healing compositions of Embodiment One (I.A-D)with women's hygeine products with and without drugs or antimicrobialswould provide an augmented wound healing composition (I.A-D+M) whichwould heal the irritated skin and tissues. The augmented wound healingcomposition can be used in vaginal products with and withoutantimicrobials (reduces vaginal irritation); and post pregnancyproducts, i.e., breast and stomach creams (reduces stretch marks).

In a specific embodiment, the invention is directed to an augmentedwound healing composition (Embodiment Two (I.A-D+M)) having an enhancedability to prevent and reduce injury to mammalian cells which comprises:

(A) a therapeutic wound healing composition selected from the group ofconsisting of:

(I.A) (a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.B) (a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.C) (a) an antioxidant; and

(b) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.C) (a) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a medicament useful for treating injured mammalian cells.

In a preferred embodiment, the augmented wound healing compositioncomprises the therapeutic wound healing compositions of the presentinvention and a medicament useful for treating injured mammalian cellsselected from the group consisting of anti-inflammatories and woundcleansers and wound dressings. In a more preferred embodiment, theaugmented wound healing composition comprises a medicament selected fromthe group consisting of wound cleansers and wound dressings.

In a preferred embodiment, the invention is directed to an augmentedwound healing composition (I.A+M) having an enhanced ability to preventand reduce injury to mammalian cells which comprises:

(A) a therapeutic wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the resuscitation of injuredmammalian cells; and

(B) a medicament useful for treating injured mammalian cells.

B. Methods For Making the Augmented Wound Healing Compositions ofEmbodiment Two (I.A-D+M)

The present invention extends to methods for making the therapeuticaugmented wound healing compositions (I.A-D+M). In general, atherapeutic augmented wound healing composition is made by forming anadmixture of the wound healing components of Embodiment One (I.A-D) anda medicament which is useful for treating injured mammalian cells. In afirst aspect of Embodiment Two (I.A+M), an augmented wound healingtherapeutic composition is made by forming an admixture of a medicamentwhich is useful for treating injured mammalian cells and a wound healingcomposition comprising (a) a pyruvate, (b) an antioxidant, and (c) amixture of saturated and unsaturated fatty acids. In a second aspect ofEmbodiment Two (I.B+M), an augmented wound healing therapeuticcomposition is made by forming an admixture of a medicament which isuseful for treating injured mammalian cells and a wound healingcomposition comprising (a) a pyruvate, (b) a lactate, and (c) a mixtureof saturated and unsaturated fatty acids. In a third aspect ofEmbodiment Two (I.C+M), an augmented wound healing therapeuticcomposition is made by forming an admixture of a medicament which isuseful for treating injured mammalian cells and a wound healingcomposition comprising (a) an antioxidant, and (b) a mixture ofsaturated and unsaturated fatty acids. In a fourth aspect of EmbodimentTwo (I.D+M), an augmented wound healing therapeutic composition is madeby forming an admixture of a medicament which is useful for treatinginjured mammalian cells and a wound healing composition comprising (a) alactate, (b) an antioxidant, and (c) a mixture of saturated andunsaturated fatty acids.

In a preferred embodiment, the invention is directed to a method forpreparing a therapeutic augmented wound healing composition (I.A+M)which comprises the steps of admixing the following ingredients:

(A) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a therapeutically effective amount of a medicament which is usefulfor treating injured mammalian cells.

C. Methods For Employing the Augmented Wound Healing Compositions ofEmbodiment Two (I.A-D+M)

The present invention extends to methods for employing the therapeuticaugmented wound healing compositions (I.A-D+M). In general, an augmentedwound healing composition is employed by contacting the composition witha wound. In a preferred embodiment, the invention is directed to amethod for healing a wound in a mammal with an augmented wound healingcomposition (I.A+M) which comprises the steps of:

(A) providing an augmented wound healing composition which comprises:

(1) a therapeutic wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(2) a medicament useful for treating wounds; and

(B) contacting the augmented wound healing composition with the wound.

The types of wounds which may be healed using the compositions of thepresent invention are those which result from an injury which causesepidermal damage, chronic ulcers, gastric ulcers, burns, donor sitewounds, and the like. Such wounds include ophthalmic wounds, such asthose which result from corneal ulcers, radialkeratotomy, cornealtransplants, epikeratophakia and other surgically induced wounds in theeye, and cutaneous wounds such as burn wounds, donor site wounds fromskin transplants and ulcers (cutaneous, decubitus, venous stasis, anddiabetic). In addition, dermatological wounds such as psoriasis,sunburn, and skin rashes may also be treated with the compositions ofthe present invention. The compositions may be applied to the wound siteeither topically or internally depending on the type of wound.

Methods for increasing the rate of wound healing comprise contacting thewound healing composition with the wound to increase the healing rate ofthe wound. Preferably, the method comprises topically administering thecompositions of the present invention directly to a wound site. Thecomposition is maintained in contact with the wound for a period of timesufficient to increase the rate of cell growth at the wound site.

D. Formulations of the Augmented Wound Healing Compositions ofEmbodiment Two (I.A-D+M)

Once prepared, the inventive therapeutic augmented wound healingcompositions may be stored for future use or may be formulated ineffective amounts with pharmaceutically acceptable carriers such aspharmaceutical appliances and topical vehicles (oral and non-oral) toprepare a wide variety of pharmaceutical compositions. Thepharmaceutically acceptable carriers which may be employed and themethods used to prepare the pharmaceutical compositions have beendescribed above in connection with the formulations of the wound healingcompositions of Embodiment One (I.A-D).

In a specific embodiment, the invention is directed to an augmentedwound healing pharmaceutical composition which comprises:

(A) a therapeutic augmented wound healing composition which comprises:

(1) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and

(2) a therapeutically effective amount of a medicament useful fortreating wounds; andresuscitation of mammalian cells; and

(B) a pharmaceutically acceptable carrier selected from the groupconsisting of pharmaceutical appliances, bioadhesives, and occlusivevehicles.

In another specific embodiment, the invention is directed to a methodfor preparing an augmented wound healing pharmaceutical composition forincreasing the proliferation and resuscitation rate of mammalian cells,which comprises the steps of:

(A) providing a therapeutically effective amount of an augmented woundhealing composition which comprises:

(1) a medicament useful for treating wounds; and

(2) a wound healing composition comprising:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(B) providing a pharmaceutically acceptable carrier; and

(C) admixing the augmented wound healing composition from step (A) andthe pharmaceutically acceptable carrier from step (B) to form theaugmented wound healing pharmaceutical composition.

1. Immunostimulating-Wound Healing Compositions a. Embodiment Two(I.A-D+M1)

Applicant has discovered therapeutic immunostimulating-wound healingcompositions (I.A-D+M1) which comprise an immunostimulating agent (M1)and the wound healing compositions of Embodiment One (I.A-D).Preferably, the wound healing composition comprises (a) pyruvate, (b) anantioxidant, and (c) a mixture of saturated and unsaturated fatty acids.Immunostimulating agents can stimulate the immune system in a patient tokill an infecting organism but do not promote the wound healing process.Wound healing compositions can increase the resuscitation rate ofinjured mammalian cells and the proliferation rate of new mammaliancells to replace dead cells but do not kill infecting organisms.Applicants have found that the combination of an immunostimulating agentand a wound healing composition results in an immunostimulating-woundhealing composition which acts synergistically to enhance wound repairin both the upper and lower portions of the skin. The therapeuticimmunostimulating-wound healing compositions are superior in stimulatingboth the extent of re-epithelialization as well as the extent of tissueintegrity and leukocyte infiltration in the dermis.

The combination of the immunostimulating agent and the wound healingcompositions of the present invention provides a pharmaceuticalcomposition useful for healing infected wounds and having an enhancedability to prevent and reduce injury to mammalian cells and furtherincrease the resuscitation rate of injured mammalian cells. The tissuedamage associated with many infectious diseases is believed to be causedby the production of cellular produced active oxygen species.Combination of the immunostimulating agent and the wound healingcompositions may suppress such reactive oxygen-linked tissue injury.

Immunostimulating agents are compounds which stimulate the immune systemto send macrophages to the infected site. Immunostimulating agents aregenerally components of bacteria or yeast which react with rovingmacrophages to induce the immune system to respond to the foreign body.The immunostimulating agents in the immunostimulating-wound healingcompositions of the present invention may be selected from a wide rangeof therapeutic agents and mixtures of therapeutic agents. Nonlimitingillustrative categories of such immunostimulating agents includeinterleukin 1 agonists, interleukin 2 agonists, interferon agonists, RNAsynthesis inhibitors, T cell stimulating agents, and adjuvants.Adjuvants, such as Freund's complete adjuvant, are substances added toantigens to intensify the immune response to the antigen when themixture is administered to an animal. Nonlimiting illustrative specificexamples of such immunostimulating agents may be selected from the groupconsisting of betafectin™ and Freund's complete adjuvant. Preferably,the immunostimulating agent is betafectin™.

Betafectin™ (PGG-glucan, Alpha-Bert Technology, Inc. Worcester, Mass.)is a glucose polymer that stimulates and enhances specific humoral andcellular responses to challenge by infectious organisms. Betafectin™(poly(1-6)-3-D-glucopyranosyl-(1-3)-β-D-glucopyranosyl) belongs to aclass of compounds known generically as βglucans and is a highlypurified, soluble, active molecule derived from a nonrecombinant yeaststrain of Saccharomyces cerevisiae. Betafectin™ lacks in vivo pyrogenicand inflammatory effects resulting from cytokine induction, but retainspotent immunostimulatory properties. Betafectin™ has a high affinity forβ-glucan receptors of human monocytes and neutrophils and bindscompetitively to the receptor in a dose-dependent manner atconcentrations significantly below those required for other naturalglucan preparations derived from bakers' yeast. Betafectin™significantly increases human neutrophils and macrophage in vitromicrobicidal activity against Staphylococcus aureus without directlystimulating synthesis of the cytokines, interleukin-1, or tumor necrosisfactor.

β-Glucans are complex polysaccharides composed entirely of glucosesubunits. When polymerized, glucose can form a wide array ofmacromolecular structures with dramatically different biologicfunctions. For example, when glucose subunits are bound together betweenthe 1 and the 4 positions, cellulose is formed, but a 1 to 3 connectionresults in the formation of β-glucans. Glucose polymers are not alwaysstraight chain molecules. In the case of β-glucan, short branches offthe main backbone are also found. As extracted from the yeast, β-glucansare not very soluble in water. In an aqueous environment, they willexist in a mixture of configurations ranging from random coils toaggregates of triple helices. Betafectin™ is not an activator but rathera primer of the immune response. When a cell is primed, the cell ispushed into a state whereby it is ready to respond when an activator ispresent but the primer itself does not induce any obvious change in thecell's behavior.

The amount of immunostimulating agent used in the present invention mayvary depending upon the therapeutic dosage recommended or permitted forthe particular immunostimulating agent. In general, the amount ofimmunostimulating agent present is the ordinary dosage required toobtain the desired result. Such dosages are known to the skilledpractitioner in the medical arts and are not a part of the presentinvention. In a preferred embodiment, the immunostimulating agent in theimmunostimulating-wound healing composition is present in an amount fromabout 0.001% to about 10%, preferably from about 0.01% to about 1%, andmore preferably from about 0.1% to about 0.5%, by weight.

b. Methods For Making the Immunostimulating-Wound Healing Compositionsof Embodiment Two (I.A-D+M1)

The present invention extends to methods for making the therapeuticimmunostimulating-wound healing compositions (I.A-D+M1). In general, atherapeutic immunostimulating-wound healing composition is made byforming an admixture of the wound healing components of Embodiment One(I.A-D) and an immunostimulating agent. In a first aspect of EmbodimentTwo (I.A+M1), an immunostimulating-wound healing therapeutic compositionis made by forming an admixture of an immunostimulating agent and awound healing composition I.A, comprising (a) a pyruvate, (b) anantioxidant, and (c) a mixture of saturated and unsaturated fatty acids.In a second aspect of Embodiment Two (I.B+M1), animmunostimulating-wound healing therapeutic composition is made byforming an admixture of an immunostimulating agent and a wound healingcomposition I.B, comprising (a) a pyruvate, (b) a lactate, and (c) amixture of saturated and unsaturated fatty acids. In a third aspect ofEmbodiment Two (I.C+M1), an immunostimulating-wound healing therapeuticcomposition is made by forming an admixture of an immunostimulatingagent and a wound healing composition I.C, comprising (a) anantioxidant, and (b) a mixture of saturated and unsaturated fatty acids.In a fourth aspect of Embodiment Two (I.D+M1), animmunostimulating-wound healing therapeutic composition is made byforming an admixture of an immunostimulating agent and a wound healingcomposition I.D, comprising (a) a lactate, (b) an antioxidant, and (c) amixture of saturated and unsaturated fatty acids.

In a preferred embodiment, the invention is directed to a method forpreparing a therapeutic immunostimulating-wound healing composition(I.A+M1) which comprises the steps of admixing the followingingredients:

(A) a therapeutically effective amount of an immunostimulating agent;and

(B) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

c. Methods For Employing the Immunostimulating-Wound HealingCompositions of Embodiment Two (I.A-D+M1)

The present invention extends to methods for employing the therapeuticimmunostimulating-wound healing compositions (I.A-D+M1). In general, atherapeutic composition is employed by contacting the therapeuticcomposition with a wound. In a preferred embodiment, the invention isdirected to a method for healing an infected wound in a mammal with animmunostimulating-wound healing composition (I.A+M1) which comprises thesteps of:

(A) providing a therapeutic immunostimulating-wound healing compositionwhich comprises:

(1) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and

(2) a therapeutically effective amount of an immunostimulating agent;andresuscitation of mammalian cells; and

(B) contacting the immunostimulating-wound healing composition with theinfected wound.

d. Augmented Immunostimulating-Wound Healing Compositions of EmbodimentTwo (I.A-D+M1+M)

In another aspect of Embodiment Two, the immunostimulating-wound healingcompositions (I.A-D+M1) of the present invention may be further combinedwith medicaments useful for treating wounds (M) to form augmentedimmunostimulating-wound healing compositions (I.A-D+M1+M). In thisembodiment, the combination of the immunostimulating-wound healingcomposition of the present invention and the medicament useful fortreating wounds provides an augmented immunostimulating-wound healingcomposition having an enhanced ability to increase the proliferation andresuscitation rate of mammalian cells. For example, the therapeuticcompositions of the present invention may be used in combination withmedicaments useful for treating wounds such as other immunostimulatingagents (Betafectin™), antiviral agents, antikeratolytic agents,anti-inflammatory agents, antifungal agents, tretinoin, sunscreenagents, dermatological agents, topical antihistamine agents,antibacterial agents, bioadhesive agents, respiratory burstinginhibitors (lactic acid, adenosine), inhibitors of prostaglandinsynthesis (ibuprofen, aspirin, indomethacin, meclofenomic acid, retinoicacid, padimate O, meclomen, oxybenzone), steroidal anti-inflammatoryagents (corticosteroids including synthetic analogs), antimicrobialagents (neosporin ointment, silvadine), antiseptic agents, anestheticagents (pramoxine hydrochloride, lidocaine, benzocaine), cell nutrientmedia, burn relief medications, sun burn medications, acne preparations,insect bite and sting medications, wound cleansers, wound dressings,scar reducing agents (vitamin E), and the like, and mixtures thereof, tofurther enhance the proliferation and resuscitation rate of mammaliancells. Preferably, the medicament useful for treating wounds is selectedfrom the group consisting of immunostimulating agents, antiviral agents,antikeratolytic agents, anti-inflammatory agents, antifungal agents,tretinoin, sunscreen agents, dermatological agents, topicalantihistamine agents, antibacterial agents, bioadhesive agents,respiratory bursting inhibitors, inhibitors of prostaglandin synthesis,antimicrobial agents, cell nutrient media, scar reducing agents, andmixtures thereof. More preferably, the medicament useful for treatingwounds is selected from the group consisting of immunostimulatingagents, antiviral agents, antikeratolytic agents, anti-inflammatoryagents, antifungal agents, acne treating agents, sunscreen agents,dermatological agents, antihistamine agents, antibacterial agents,bioadhesive agents, and mixtures thereof.

In a preferred embodiment, the invention is directed to an augmentedimmunostimulating-wound healing composition (I.A+M1+M) which comprises:

(A) a therapeutic immunostimulating-wound healing composition whichcomprises:

(1) a therapeutically effective amount of an immunostimulating agent;and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a medicament useful for treating wounds.

The present invention extends to methods for making the augmentedimmunostimulating-wound healing compositions. In general, the augmentedcompositions are made by admixing the therapeuticimmunostimulating-wound healing composition with the medicament usefulfor treating wounds to prepare the augmented immunostimulating-woundhealing composition.

The present invention also extends to methods for employing theaugmented immunostimulating-wound healing compositions. In general, anaugmented immunostimulating-wound healing composition is employed bycontacting the composition with a wound. In a preferred embodiment, theinvention is directed to a method for healing an infected wound in amammal with an augmented immunostimulating-wound healing composition(I.A+M1+M) which comprises the steps of:

(A) providing a therapeutic augmented immunostimulating-wound healingcomposition which comprises:

(1) a therapeutically effective amount of an immunostimulating agent;

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(3) providing a medicament useful for treating wounds; and

(B) contacting the augmented immunostimulating-wound healing compositionwith the infected wound.

The types of wounds which may be healed using theimmunostimulating-wound healing compositions and the augmentedimmunostimulating-wound healing compositions of the present inventionare those which result from an infected injury which causes epidermaldamage. The topical therapeutic compositions may be used orally in theform of a mouth wash or spray to protect and accelerate the healing ofinjured oral tissue.

Methods for healing a wound comprise topically administering thecompositions of the present invention directly to a wound site toincrease the healing rate of the wound. The composition is maintained incontact with the wound for a period of time sufficient to increase theproliferation and resuscitation rate of the cells.

e. Formulations of the Immunostimulating-Wound Healing Compositions ofEmbodiment Two (I.A-D+M1) and (I.A-D+MI+M)

Once prepared, the inventive therapeutic immunostimulating-wound healingcompositions and augmented immunostimulating-wound healing compositionsmay be stored for future use or may be formulated in effective amountswith pharmaceutically acceptable carriers such as pharmaceuticalappliances and topical vehicles (oral and non-oral) to prepare a widevariety of pharmaceutical compositions. The pharmaceutically acceptablecarriers which may be employed and the methods used to prepare thepharmaceutical compositions have been described above in connection withthe formulations of the wound healing compositions of Embodiment One(I.A-D).

In a preferred embodiment, the invention is directed to animmunostimulating-wound healing pharmaceutical composition whichcomprises:

(A) a therapeutic immunostimulating-wound healing composition (I.A+M1)which comprises:

(1) a therapeutically effective amount of an immunostimulating agent;and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a pharmaceutically acceptable carrier selected from the groupconsisting of pharmaceutical appliances, bioadhesives, and occlusivevehicles.

In another preferred embodiment, the invention is directed to a methodfor preparing a pharmaceutical composition for increasing theproliferation and resuscitation rate of mammalian cells, which comprisesthe steps of:

(A) providing a therapeutically effective amount of animmunostimulating-wound healing composition (I.A+M1) which comprises:

(1) an immunostimulating agent; and

(2) a wound healing composition comprising:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(B) providing a pharmaceutically acceptable carrier; and

(C) admixing the immunostimulating-wound healing composition from step(A) and the pharmaceutically acceptable carrier from step (B) to form apharmaceutical composition.

f. Examples of the Immunostimulating-Wound Healing Compositions ofEmbodiment Two (I.A-D+M1) Study 1

This study demonstrates a comparison of the wound healing abilities ofthe therapeutic wound healing compositions of the present invention ininfected and noninfected mice versus conventional wound healingcompositions. The results of this study are illustrated in examples A-I.

The wound healing studies were carried out using hairless mice. Fiveformulations were examined in a randomized double blind study. Therewere 6 mice in each study group for evaluation at either day three orday seven. A midline, 3 cm, full thickness longitudinal incision wasmade on the anesthetized mice. The creams were applied 2 hours followingthe wounding and reapplied at 24 hour intervals during the 7 day study.Betafectin™, the immunostimulating agent, was applied 20 minutes beforethe application of the creams. Wounds were examined daily and scored forclosure on a scale of 0-5 with 5 indicating the most healed. The animalswere sacrificed and the tissue samples were examined histologically.Both infected and noninfected wounds were used. Regardless of thepresence of infection, the compositions with Betafectin™ with live yeastcell derivative (LYCD) and the wound healing composition weresignificantly better as wound healing agents then the other testedformulas. This wound healing ability was demonstrated by subjectivegrading of healing through wound closure and objective histologicalexaminations of tissue sections. The order for wound healing efficacyfollowing the Betafectin™ with live yeast cell derivative and woundhealing composition was as follows: 2) Betafectin; (3) wound healingcomposition with live yeast cell derivative; 4) Neosporin and woundhealing composition; and 5) untreated control.

A detailed summary of studies is provided below that were performed toestablish that formulations including Betafectin™, the immunostimulatingagent, alone and in combination with formulations including PreparationH™ or a Neosporin base with live yeast cell derivative (LYCD) and thewound healing composition of compounds, sodium pyruvate, Vitamin E(alpha-tocopherol) and fatty acids enhance wound healing.

There were 9 groups of mice, with N=6 per group, and the groups had thefollowing treatment: A) wounded, no treatment (control); B) Betafectin™alone, 2 μg/mouse per day, added topically in Phosphate Buffered Saline(PBS); C) Preparation H™ with wound healing composition; D) PreparationH™ with Betafectin™; and E) Neosporin formulation with wound healingcomposition. Groups F-I consisted of groups of 6 mice each that weretreated with a culture of 10⁷ Staphylococcus 30 minutes after woundingand 2 hours prior to treatment with any formulation. Groups F-Iconstituted the infection model to determine the ability of Betafectin™as well as the Neosporin formulation to enhance wound healing in thepresence of an infection.

Studies were carried out using hairless SKH-1 inbred mice, 6-8 weeks ofage. Mice were placed in cages of 6 each. Mice were anesthetized withether and a midline 3 cm full thickness longitudinal incision was madewith a number 10 scalpel blade, producing a full thickness wound thatdid not penetrate the underlying fascia. Incisions were closed usingsteel clips at 1 centimeter intervals. Formulations were applied 30minutes following wounding and reapplied at 24 hours intervals duringthe 7 day post-operative period. Wounds were examined daily and rankordered for effectiveness of formulation for enhancement of visual basedwound healing each day.

Animals were photographed at day 3 or 4 of the experimental protocol fordocumentation of visible effects of the treatments. Animals weresacrificed using ether euthanasia on day 3 and day 7. The dorsal skinincluding the incision was dissected without the subcutaneous tissue.The skin was placed in neutral buffered formalin and subsequentlysectioned and stained with hematoxylin and eosin. Microscopicexamination of the wounds was performed and representative tissuesectioned photographed.

The Treatment Groups were as follows:

A. Control-wounded, no treatment.

B. Freshly prepared Preparation H™ formulation containing live yeastcell derivative (LYCD) and the wound healing composition of sodiumpyruvate, Vitamin E, and 1% fatty acids.

C. Neosporin formulation with the wound healing composition.

D. Betafectin™ in a solution of Phosphate Buffered Saline, addedtopically to the wound.

E. Betafectin™ and Preparation H™ with the wound healing composition.The Betafectin™ was added topically in an aqueous solution 20 minutesprior to the application of the formulation with wound healingcomposition and live yeast cell derivative.

F-I. These groups were treated as set out above, but with the additionof 107 Staphylococcus aureus as a bacterial infection agent applied 2hours prior to the formulations to be tested.

Photographs of the wounded mice are set out in FIGS. 11A-14.

FIGS. 11A-11D are photographs of wounded mice after 3 days of treatmentwith: no compostion (FIG. 11A); Betafectin™ with Preparation H™ with thewound healing composition (FIG. 11B); Betafectin™ (FIG. 11C); live yeastcell derivative containing the wound healing composition (FIG. 11D).

FIG. 12 is a photograph of a wounded mouse after 3 days of treatmentwith Neosporin™ containing the wound healing composition.

FIGS. 13A-13D are photographs of the histological results of woundedmice after 3 days of treatment with: no composition (FIG. 13A);Betafectin™ with Preparation H™ with the wounded healing composition)FIG. 13B); Betafection™ (FIG. 13C); live yeast cell derivativecontaining the wound healing composition (FIG. 13D).

FIG. 14 is a photograph of the histological results of a wounded mouseafter 3 days of treatment with neosporin containing the wound healingcomposition (D).

Results: Regardless of the presence of infection, the rank order forwound healing efficacy (wound healing and closure, as well ashistological correlation of rate of wound healing) was:

    (E) wound healing composition and Betafectin™>(D) Betafectin™>(B) wound healing composition=(C) Neosporin>>Untreated.

Histological Description of Wounded Tissue at Day 3 From Each of theTreatment Groups

A) Control: Wounded-No Treatment: Lower portion of the wounded area,i.e., dermis has heavy infiltration with both lymphocytes andmonocytes/macrophages. The re-epithelialization that occurs at the outermost layer of the skin, the epidermal layer, is not complete. The tissuesection shows that the dermal tissue is weak, in that the tissueintegrity was not maintained when it was sectioned.

B) Preparation H™ formulation with wound healing composition: From thethicker outer layer of the epidermis, the re-epithelialization processhas progressed further than was apparent in the untreated controlwounded tissue. Although there is infiltration of the dermis withmonocytes and lymphocytes, it does not appear to be as diffuse or asheavy as with the control untreated wound tissue.

C) Neosporin formulation with the wound healing composition: The resultswith this formulation show that the re-epithelialization was extensiveand rapid. In contrast, the infiltration of the macrophages andlymphocytes within the dermis was less than observed in either thecontrol untreated wound tissue or the wound tissue treated with thePreparation H™ containing the wound healing composition combination ofagents. It appears that the formulations containing the combination ofVitamin E, sodium pyruvate, and fatty acids stimulates proliferation ofthe outer layer of the epidermis.

D) Betafectin™ in phosphate buffered saline: The histology of thewounded tissue isolated from mice treated for 3 days with Betafectin™show that there is a heavy infiltration of macrophages and lymphocytesand that there is apparently greater integrity of the tissue than thatobserved with any of the other treatments. The re-epithelialization isnot, however, comparable to that observed with either the Neosporin orthe Preparation H™ formulations that contain the wound healingcomposition.

E) Betafectin™ and Preparation H™ wound healing composition formulation:The histological sections isolated from tissue taken from mice treatedwith this formulation was by far superior in terms of both the extent ofre-epithelialization as well as the extent of tissue integrity andleukocyte infiltration in the lower portion of the skin, the dermis. Itis apparent that this combination of ingredients is able to actsynergistically to enhance wound repair in both the upper and lowerportions of the skin.

The combination of Betafectin™ and Preparation H™ wound healingcomposition stimulated both an increase in wound healing within thelower portion of the wound, within the dermal layer, as well asstimulated the re-epithelialization of the outer layer of the wound.These two activities resulted in a rapidly healed wound at day 3following wounding. The difference in the activities of the otherformulations is because complete and rapid healing of wounds requiresstimulation of healing in the outer and lower areas of the skin. Fromhistological examination of the wounds, it is apparent that both theBetafectin™ alone as well as the wound healing composition alonestimulated the dermal area for wound healing. In contrast, the Neosporinformulation stimulated the re-epithelialization, that is stimulated theouter components of the skin to enhance wound closure. The combinationof Betafectin™ with the wound healing composition in a Preparation H™like formulation resulted in a synergistic enhancement of wound healingthat was significantly better than any of the agents alone.

The mice that were infected with S. aureus and not treated with anyagents were sacrificed at day 3, since the infection was not treatedwith antibiotics and remained purulent. The histology of tissue isolatedfrom wounds that were treated with formulations of Betafectin™,Preparation H™, and wound healing composition or neosporin and woundhealing composition, did not appear to differ with any of the treatmentsexcept for the presence of polymorphonuclear cells within the dermis,where in normal uninfected tissue, the predominant cell type wasmonocytes and lymphocytes.

In summary, these studies show that there are several componentsnecessary to prepare an effective formulation to enhance wound healing:(1) enhance the activity of the epithelial cells within the outer layerof the skin so that re-epithelialization occurs at a rapid and efficientmanner;, (2) stimulate the activity of the inflammatory and immune cellsthat come into the lower part of the skin, the dermis, in the earlyportion of the wound healing process; (3) prepare a stable formulationthat is compatible with the Betafectin™ and the wound healingcomposition combination with live yeast cell derivative.

2. Antiviral-Wound Healing Compositions a. Embodiment Two (I.A-D+M2)

Applicant has discovered therapeutic antiviral-wound healingcompositions (I.A-D+M2) which comprise an antiviral agent (M2) and thewound healing compositions of Embodiment One (I.A-D). Preferably, thewound healing composition (I.A) comprises (a) pyruvate, (b) anantioxidant, and (c) a mixture of saturated and unsaturated fatty acids.Antiviral agents can reduce virus titers in a patient but do not promotethe wound healing process. Wound healing compositions can increase theresuscitation rate of injured mammalian cells and the proliferation rateof new mammalian cells to replace dead cells but do not reduce virustiters. Applicants have found that the combination of an antiviral agentand a wound healing composition results in a therapeutic antiviral-woundhealing composition which reduces the size, duration, and severity oforal and vaginal wounds suffered from viruses such as herpes.

The combination of the antiviral agent and the wound healingcompositions of the present invention provides a pharmaceuticalcomposition useful for reducing virus titers and having an enhancedability to prevent and reduce injury to mammalian cells and furtherincrease the resuscitation rate of injured mammalian cells. The tissuedamage associated with many viral diseases is believed to be caused bythe production of cellular produced active oxygen species. Combinationof the antiviral agent and the wound healing compositions may suppresssuch reactive oxygen-linked tissue injury.

The antiviral agents in the antiviral-wound healing compositions of thepresent invention may be selected from a wide variety of water-solubleand water-insoluble drugs and their acid addition or metallic salts.Both organic and inorganic salts may be used provided the antiviralagent maintains its medicament value. The antiviral agents may beselected from a wide range of therapeutic agents and mixtures oftherapeutic agents which may be administered in sustained release orprolonged action form. Nonlimiting illustrative categories of suchantiviral agents include RNA synthesis inhibitors, protein synthesisinhibitors, immunostimulating agents, protease inhibitors, andcytokines. Nonlimiting illustrative specific examples of such antiviralagents include the following medicaments.

(a) Acyclovir (9-[(2-hydroxyethyloxy)methyl]guanine, tradename--Zovirax™) is an antiviral drug for oral administration. Acycloviris a white, crystalline powder with a molecular weight of 225 daltonsand a maximum solubility in water of 2.5 mg/mL at 37° C. Acyclovir is asynthetic purine nucleoside analogue with in vitro and in vivoinhibitory activity against human herpes viruses including herpessimplex types 1 (HSV-1) and 2 (HSV-2), varicella-zoster virus (VZV),Epstein-Barr virus (EBV), and cytomegalovirus (CMV).

(b) Foscarnet sodium (phosphonoformic acid trisodium salt, tradename--Foscavir™) is an antiviral drug for intravenous administration.Foscarnet sodium is a white, crystalline powder containing 6 equivalentsof water of hydration with an empirical formula of Na₃ CO₆ P.6 H₂ O anda molecular weight of 300.1. Foscarnet sodium has the potential tochelate divalent metal ions such as calcium and magnesium, to formstable coordination compounds. Foscarnet sodium is an organic analogueof inorganic pyrophosphate that inhibits replication of all known herpesviruses in vitro including cytomegalovirus (CMV), herpes simplex virustypes 1 and 2 (HSV-1, HSV-2), human herpes virus 6 (HHV-6), Epstein-Barrvirus (EBV), and varicella-zoster virus (VZV). Foscarnet sodium exertsits antiviral activity by a selective inhibition at the pyrophosphntebinding site on virus-specific DNA polymerases and reversetranscriptases at concentrations that do not affect cellular DNApolymerases.

(c) Ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide,trade name--Virazole™) is an antiviral drug provided as a sterile,lyophilized powder to be reconstituted for aerosol administration.Ribavirin is a synthetic nucleoside which is a stable, white,crystalline compound with a maximum solubility in water of 142 mg/ml at25° C. and with only a slight solubility in ethanol. The empiricalformula is C₈ H₁₂ N₄ O₅ and the molecular weight is 244.2 Daltons.Ribavirin has antiviral inhibitory activity in vitro against respiratorysyncytial virus, influenza virus, and herpes simplex virus. Ribavirin isalso active against respiratory syncytial virus (RSV) in experimentallyinfected cotton rats. In cell cultures, the inhibitory activity ofribavirin for RSV is selective. The mechanism of action is unknown.Reversal of the in vitro antiviral activity by guanosine or xanthosinesuggests ribavirin may act as an analogue of these cellular metabolites.

(d) Vidarabine (adenine arabinoside, Ara-A,9-β-D-arabinofuranosyladenine monohydrate, trade name--Vira-A™) is anantiviral drug. Vidarabine is a purine nucleoside obtained fromfermentation cultures of Streptomyces antibioticus. Vidarabine is awhite, crystalline solid with the empirical formula, C₁₀ H₁₃ N₅ O₄.H₂ O.The molecular weight of vidarabine is 285.2, the solubility is 0.45mg/ml at 25° C., and the melting point ranges from 260° to 270° C.Vidarabine possesses in vitro and in vivo antiviral activity againstHerpes simplex virus types 1 and 2 (HSV-1 and HSV-2), and in vitroactivity against varicella-zoster virus (VZV). The antiviral mechanismof action has not yet been established. Vidarabine is converted intonucleotides which inhibit viral DNA polymerase.

(e) Ganeiclovir sodium (9-(1,3-dihydroxy-2-propoxymethyl)guanine,monosodium salt, trade name--Cytovene™) is an antiviral drug activeagainst cytomegalovirus for intravenous administration. Ganeiclovirsodium has a molecular formula of C₉ H₁₂ N₆ NaO₄ and a molecular weightof 277.21. Ganeiclovir sodium is a white lyophilized powder with anaqueous solubility of greater than 50 mg/mL at 25° C. Ganeiclovir is asynthetic nucleoside analogue of 2'-deoxyguanosine that inhibitsreplication of herpes viruses both in vitro and in vivo. Sensitive humanviruses include cytomegalovirus (CMV), herpes simplex virus-1 and -2(HSV-1, HSV-2), Epstein-Barr virus (EBV), and varicella zoster virus(VZV).

(f) Zidovudine [azidothymidine (AZT), 3'-azido-3'-deoxythymidine, tradename--Retrovir™] is an antiretroviral drug active against humanimmunodeficiency virus (HIV) for oral administration. Zidovudine is awhite to beige, odorless, crystalline solid with a molecular weight of267.24 daltons and a molecular formula of C₁₀ H₁₃ N₅ O₄. Zidovudine isan inhibitor of the in vitro replication of some retroviruses includingHIV (also known as HTLV III, LAV, or ARV). Zidovudine is a thymidineanalogue in which the 3'hydroxy (--OH) group is replaced by an azido(--N3) group.

(g) Phenol (carbolic acid) is a topical antiviral, anesthetic,antiseptic, and antipruritic drug. Phenol is a colorless or whitecrystalline mass which is soluble in water, has a characteristic odor, amolecular formula of C₆ H₆ O, and a molecular weight of 94.11.

(h) Amantadine hydrochloride (1-adamantanamine hydrochloride, tradename--Symmetrel™) has pharmacological actions as both an anti-Parkinsonand an antiviral drug. Amantadine hydrochloride is a stable white ornearly, white crystalline powder, freely soluble in water and soluble inalcohol and in chloroform. The antiviral activity of amantadinehydrochloride against influenza A is not completely understood but themode of action appears to be the prevention of the release of infectiousviral nucleic acid into the host cell.

(i) Interferon alfa-n3 (human leukocyte derived, trade name--Alferon™)is a sterile aqueous formulation of purified, natural, human interferonalpha proteins for use by injection. Interferon alfa-n3 injectionconsists of interferon alpha proteins comprising approximately 166 aminoacids ranging in molecular weights from 16,000 to 27,000 daltons.Interferons are naturally occurring proteins with both antiviral andantiproliferative properties.

Preferred antiviral agents to be employed may be selected from the groupconsisting of acyclovir, foscarnet sodium, ribavirin, vidarabine,ganeiclovir sodium, zidovudine, phenol, amantadine hydrochloride, andinterferon alfa-n3. In a preferred embodiment, the antiviral agent isselected from the group consisting of acyclovir, foscarnet sodium,ribavirin, vidarabine, and ganeiclovir sodium. In a more preferredembodiment, the antiviral agent is acyclovir.

The antiviral agent of the present invention may be used in manydistinct physical forms well known in the pharmaceutical art to providean initial dosage of the antiviral agent and/or a further time-releaseform of the antiviral agent. Without being limited thereto, suchphysical forms include free forms and encapsulated forms, and mixturesthereof.

The amount of antiviral agent used in the present invention may varydepending upon the therapeutic dosage recommended or permitted for theparticular antiviral agent. In general, the amount of antiviral agentpresent is the ordinary dosage required to obtain the desired result.Such dosages are known to the skilled practitioner in the medical artsand are not a part of the present invention. In a preferred embodiment,the antiviral agent in the antiviral-wound healing composition ispresent in an amount from about 0.1% to about 20%, preferably from about1% to about 10%, and more preferably from about 2% to about 7%, byweight.

b. Methods For Making the Antiviral-Wound Healing Compositions ofEmbodiment Two (I.A-D+M2)

The present invention extends to methods for making the therapeuticantiviral-wound healing compositions (I.A-D+M2). In general, atherapeutic antiviral-wound healing composition is made by forming anadmixture of the wound healing components of Embodiment One (I.A-D) andan antiviral agent. In a first aspect of Embodiment Two (I.A+M2), anantiviral-wound healing therapeutic composition is made by forming anadmixture of an antiviral agent and a wound healing compositioncomprising (a) a pyruvate, (b) an antioxidant, and (c) a mixture ofsaturated and unsaturated fatty acids. In a second aspect of EmbodimentTwo (I.B+M2), an antiviral-wound healing therapeutic composition is madeby forming an admixture of an antiviral agent and a wound healingcomposition comprising (a) a pyruvate, (b) a lactate, and (c) a mixtureof saturated and unsaturated fatty acids. In a third aspect ofEmbodiment Two (I.C+M2), an antiviral-wound healing therapeuticcomposition is made by forming an admixture of an antiviral agent and awound healing composition comprising (a) an antioxidant, and (b) amixture of saturated and unsaturated fatty acids. In a fourth aspect ofEmbodiment Two (I.D+M2), an antiviral-wound healing therapeuticcomposition is made by forming an admixture of an antiviral agent and awound healing composition comprising (a) a lactate, (b) an antioxidant,and (c) a mixture of saturated and unsaturated fatty acids.

In a preferred embodiment, the invention is directed to a method forpreparing a therapeutic antiviral-wound healing composition (I.A+M2)which comprises the steps of admixing the following ingredients:

(A) a therapeutically effective amount of an antiviral agent; and

(B) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

c. Methods For Employing the Antiviral-Wound Healing Compositions ofEmbodiment Two (I.A-D+M2)

The present invention extends to methods for employing the therapeuticantiviral-wound healing compositions (I.A-D+M2). In general, atherapeutic composition is employed by contacting the therapeuticcomposition with a wound. In a preferred embodiment, the invention isdirected to a method for healing an infected wound in a mammal with anantiviral-wound healing composition (I.A+M2) which comprises the stepsof:

(A) providing a therapeutic antiviral-wound healing composition whichcomprises:

(1) a therapeutically effective amount of an antiviral agent; and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) contacting the antiviral-wound healing composition with the infectedwound.

d. The Antiviral-Wound Healing Compositions of Embodiment Two(I.A-D+M2+M)

In another aspect of Embodiment Two, the therapeutic antiviral-woundhealing compositions (I.A-D+M2) of the present invention may be furthercombined with medicaments useful for treating wounds (M) to formaugmented antiviral-wound healing compositions (I.A-D+M2+M). In thisembodiment, the combination of the antiviral-wound healing compositionof the present invention and the medicament useful for treating woundsprovides an augmented antiviral-wound healing composition having anenhanced ability to increase the proliferation and resuscitation rate ofmammalian cells. For example, the therapeutic compositions of thepresent invention may be used in combination with medicaments useful fortreating wounds such as immunostimulating agents (Betafectin™), otherantiviral agents, antikeratolytic agents, anti-inflammatory agents,antifungal agents, tretinoin, sunscreen agents, dermatological agents,topical antihistamine agents, antibacterial agents, bioadhesive agents,respiratory bursting inhibitors (lactic acid, adenosine), inhibitors ofprostaglandin synthesis (ibuprofen, aspirin, indomethacin, meclofenomicacid, retinoic acid, padimate O, meclomen, oxybenzone), steroidalanti-inflammatory agents (corticosteroids including synthetic analogs),antimicrobial agents (neosporin ointment, silvadine), antiseptic agents,anesthetic agents (pramoxine hydrochloride, lidocaine, benzocaine), cellnutrient media, burn relief medications, sun burn medications, acnepreparations, insect bite and sting medications, wound cleansers, wounddressings, scar reducing agents (vitamin E), and the like, and mixturesthereof, to further enhance the proliferation and resuscitation rate ofmammalian cells. Preferably, the medicament useful for treating woundsis selected from the group consisting of immunostimulating agents,antiviral agents, antikeratolytic agents, anti-inflammatory agents,antifungal agents, tretinoin, sunscreen agents, dermatological agents,topical antihistamine agents, antibacterial agents, bioadhesive agents,respiratory bursting inhibitors, inhibitors of prostaglandin synthesis,antimicrobial agents, cell nutrient media, scar reducing agents, andmixtures thereof. More preferably, the medicament useful for treatingwounds is selected from the group consisting of immunostimulatingagents, antiviral agents, antikeratolytic agents, anti-inflammatoryagents, antifungal agents, acne treating agents, sunscreen agents,dermatological agents, antihistamine agents, antibacterial agents,bioadhesive agents, and mixtures thereof.

In a preferred embodiment, the invention is directed to an augmentedantiviral-wound healing composition (I.A+M2+M) which comprises:

(A) a therapeutic antiviral-wound healing composition which comprises:

(1) a therapeutically effective amount of an antiviral agent; and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a medicament useful for treating wounds.

The present invention extends to methods for making the augmentedantiviral-wound healing compositions. In general, the augmentedcompositions are made by admixing the therapeutic antiviral-woundhealing composition with the medicament useful for treating wounds toprepare the augmented antiviral-wound healing composition.

The present invention also extends to methods for employing theaugmented antiviral-wound healing compositions. In general, an augmentedantiviral-wound healing composition is employed by contacting thecomposition with a wound. In a preferred embodiment, the invention isdirected to a method for healing an infected wound in a mammal with anaugmented antiviral-wound healing composition (I.A+M2+M) which comprisesthe steps of:

(A) providing a therapeutic augmented antiviral-wound healingcomposition which comprises:

(1) a therapeutically effective amount of an antiviral agent;

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(3) providing a medicament useful for treating wounds; and

(B) contacting the augmented antiviral-wound healing composition withthe infected wound.

The types of wounds which may be healed using the antiviral-woundhealing compositions and the augmented antiviral-wound healingcompositions of the present invention are those which result from aninfected injury which causes epidermal damage. The topical therapeuticcompositions may be used orally in the form of a mouth wash or spray toprotect and accelerate the healing of injured oral tissue.

Methods for healing a wound comprise topically administering thecompositions of the present invention directly to a wound site toincrease the healing rate of the wound. The composition is maintained incontact with the wound for a period of time sufficient to increase theproliferation and resuscitation rate of the cells.

e. Formulations of the Antiviral-Wound Healing Compositions ofEmbodiment Two (I.A-D+M2) and (I.A-D+M2+M)

Once prepared, the inventive therapeutic antiviral-wound healingcompositions and augmented antiviral-wound healing compositions may bestored for future use or may be formulated in effective amounts withpharmaceutically acceptable carriers such as pharmaceutical appliancesand topical vehicles (oral and non-oral) to prepare a wide variety ofpharmaceutical compositions. The pharmaceutically acceptable carrierswhich may be employed and the methods used to prepare the pharmaceuticalcompositions have been described above in connection with theformulations of the wound healing compositions of Embodiment One(I.A-D).

In a preferred embodiment, the invention is directed to anantiviral-wound healing pharmaceutical composition which comprises:

(A) a therapeutic antiviral-wound healing composition (I.A+M2) whichcomprises:

(1) a therapeutically effective amount of an antiviral agent; and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a pharmaceutically acceptable carrier selected from the groupconsisting of pharmaceutical appliances, bioadhesives, and occlusivevehicles.

In another preferred embodiment, the invention is directed to a methodfor preparing a pharmaceutical composition for increasing theproliferation and resuscitation rate of mammalian cells, which comprisesthe steps of:

(A) providing a therapeutically effective amount of an antiviral-woundhealing composition (I.A+M2) which comprises:

(1) an antiviral agent; and

(2) a wound healing composition comprising:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(B) providing a pharmaceutically acceptable carrier; and

(C) admixing the antiviral-wound healing composition from step (A) andthe pharmaceutically acceptable carrier from step (B) to form apharmaceutical composition.

f. Examples of the Antiviral-Wound Healing Compositions of EmbodimentTwo (I.A-D+M2) Study 1

This study demonstrates a comparison of the wound healing abilities ofthe therapeutic antiviral-wound healing compositions of the presentinvention versus conventional wound healing compositions. The results ofthis study are illustrated in examples 1-21.

Two animal models were used to examine the ability of the wound healingcomponents to reduce lesion development, duration, and severity.Mathematical modeling was used to determine the ratio and concentrationsof wound healing components used in the animal models. In the guinea pigmodel, formulas #11 and #17 reduced lesion development, duration, andseverity scores significantly compared to the vehicle control, Blistex™,and Acyclovir. Acyclovir was the only compound that reduced viral titerssignificantly. In the mouse model, formulas #1, #15, and #16 reducedclinical symptoms compared to the vehicle and Blistex™. Acyclovir, inthe mouse model, reduced lesion development, duration and severity,producing the best results. Statistical analysis of the data haveconfirmed the results. In both models, the best formulas contain anequal ratio of Vitamin E and pyruvate and the higher levels of fattyacids. In the guinea pig model, formulas #11 and #17 contained 0.5% ofboth Vitamin E and pyruvate. In the mouse model, formulas #1 and #16contained 4.75% of the same actives. Deviation from these ratios reducedthe herpes lesion healing efficacy for both models.

Guinea Pig Studies

The purpose of these studies was to evaluate the activity of variousantioxidant preparations administered topically in a primary genitalHSV-2 infection of guinea pigs. Eighteen different preparationscontaining varying concentrations of three agents (vitamin E, pyruvicacid and fatty acids) were evaluated. Treatment was initiated at 48hours post-infection, which is 1-2 days before external genital lesionsbegin to appear. The commercial preparations of 5% acyclovir(ACV)-polyethylene glycol (PEG) and medicated Blistex™ for cold soreswere utilized as internal controls.

Materials and Methods

I. Medications

The experiment was placebo-controlled and the preparations were testedin a coded fashion (except for the ACV and Blistex™).

I. Genital HSV-2 Infection of Guinea Pigs

A. Description of Model

Intravaginal inoculation of weanling guinea pigs with HSV-2 results in aprimary genital infection characterized by initial replication of virusin the vaginal tract followed by the development of external vesicularlesions. Virus titers peak on days one to three in the vaginal tract andgradually clears by days 7-10. The external genital lesions first appearon day four, peak lesion severity occurs on days 6-8, and the lesionsgenerally heal by days 15-18.

B. Virus and Viral Inoculation

The MS strain of HSV-2 was utilized for animal inoculation. FemaleHartley guinea pigs (Charles River, Kingston, N.Y.) weighing 250-300 gwere inoculated intravaginally (i.vag.) with approximately 1.2×10⁵plaque forming units one hour after being swabbed for removal of vaginalsecretions. Viral inoculation was accomplished by inserting a swabsoaked with virus into the vaginal tract and rotating about six times.

C. Treatment of Guinea Pigs

Groups of 6 guinea pigs were run in duplicate, consecutive experiments(with the exception of group 21, which was run only once). The guineapigs were treated on the external genital skin with 0.1 ml of eachpreparation, four times daily for ten days beginning 48 hours post-viralinoculation.

D. Sample Collection and Virus Assays

To determine the effect of treatment on HSV-2 replication in lesions,swabs of lesions were obtained during the primary infection on days 3,4, 5, 6, 7, and 10 after HSV-2 inoculation. The swabs were placed intubes containing 2.0 ml of media, and frozen at -70° C. until titratedfor HSV. To identify the number of animals that became infected, vaginalswabs were obtained from all animals on day 5 and handled as above. Whenall samples were collected, they were thawed, vortexed, dilutedserially, and HSV-2 titers determined in rabbit kidney cells using amicrotiter CPE assay.

E. Scoring of External Lesions

To determine the effect of therapy on the development, spread, andhealing of external genital lesions, lesion severity was scored on a0-5+ scale through the primary infection (Table 7 and FIGS. 44A-48B).

F. Evaluation of Efficacy

The data for each of the two experiments were first analyzed separately,then the results were combined and reanalyzed.

Peak lesion scores, peak lesion virus titers, areas under lesionscore-day, and areas under the virus titer-day curves betweenplacebo-treated and drug-treated animals were compared using theMann-Whitney U rank sum test. A p-value of 0.05 or less was consideredsignificant.

Hairless Mouse Studies

This model was used to assess the ability of various anti-oxidantcompounds, applied topically to the infected area, to modify theclinical course of the infection.

Materials and Methods

Mice: six to eight week old male SKH-1 hairless mice (Charles River)were infected with HSV-1 virus, McIntyre strain. Infection was achievedunder general anesthesia (Ketamine, Xylazine) by the abrasion of a 1 cmsquare area (using a 25 gauge needle) centrally located on the dorsalsurface of a mouse. Virus was then applied directly onto the abradedarea (10 μl of a 1×10⁹ PFU/ml virus stock). Following inoculation of10×10⁶ PFU of HSV-1 McIntyre strain by scarification of the epidermis,herpetic lesions developed by day 5 and persisted through day 12 postinfection (p.i.). The viral lesions spread in a zosteriform pattern fromthe site of inoculation (midline on the back) to the abdominal area. Byday 10, lesions were crusted over and complete healing generallyoccurred by day 12 p.i. (post infection).

Individual mice were treated with test compounds starting on theafternoon of the infection day, and treatment was continued for 14 days.Treatments were administered at 7 a.m. and 4 p.m. each day and involvedthe use of a sterile cotton tipped applicator in such a manner that theaffected area was evenly coated with the test compound. If no lesionswere visible, only the site of infection was treated. Data, includinglesion scores, number of lesions, and lesion areas were recorded duringthe 7 a.m. treatment session. Each animal was recorded as having one offive possible scores: 0, no signs; 1, redness and swelling; 2, singlelesion; 3, multiple lesions; 4, a spread of lesions in a dermatomepattern (See FIGS. 49A-49D). In addition, the actual lesion area on theskin was measured using a micrometer (x and y axial values for eachlesion were obtained in millimeters and then multiplied together to givethe lesion area). For analysis, the individual lesions scores or areaswithin a treatment group were averaged on a daily basis.

Nineteen compounds with varying amounts of anti-oxidant compounds weretested. Control compounds included Zovirax ointment, Blistex™, andpolyethylene glycol m.w. 400. Each experimental compound was tested on atotal of eight to sixteen mice.

Statistical Evaluation Product Design/Product Group Allocation of theGuinea Pig and Mouse Models

The purpose of this study was to convey the product groups and theallocations of the animals necessary to evaluate the optimal combinationof the wound healing components (vitamin E, unsaturated fatty acids, andsodium pyruvate) in the presence of phenol and lidocaine both at 0.5% byweight. The range of the three components in the wound healing, all from0.5% to 9.0% by weight, were incorporated in the experimental design.The design is a two-cubed factorial with six star points and one centerpoint with the "control groups" Blistex™, Acyclovir and untreated. Theproduct groups are listed in a random order in the attachments (Table8). This order was random and was not changed.

The experimental variance is important for estimating the number ofanimals per product group (sample size) so that the resulting study willhave sufficient power to detect a clinically meaningful effect. Anestimate of this variance for the mouse study was obtained from therange of the clinical symptom scale. The use of eight (8) mice perproduct group should be sufficient to achieve 80% power to detect aneffect of 0.5 units when conducting a two-sided t test at the 0.05 levelof significance. The sample size for the mouse study was approximatedusing only the clinical symptoms scale. The power for detecting aclinically meaningful effect of the lesion size (total vesicular area)was not known.

The allocation of product in mice (Table 8) contains eight (8) mice perproduct group, except for the second use of Product Group Number 14which contains four (4) mice. There are two uses of Product GroupNumber 1. The numbers of the products groups in this table are the sameas those identified in Table 1 and were not changed, since the order ofthese product groups have been randomized. The side receiving the listedproduct group was also randomized. The person scoring the clinicalsymptoms and measuring the lesion size did not know which product groupwas used on which animal (blinding).

An estimate of the experimental variance for the guinea pig study wasobtained from the range of the lesion severity scale. The use of twelve(12) guinea pigs per product group should be sufficient to achieve 80%power to detect an effect of 0.5 units when conducting a two-sided ttest at the 0.05 level of significance. The sample size for the guineapig study was approximated using only the lesion severity scale. Thepowers for detecting clinically meaningful effects in the viral titer,time to healing, and other measures over time (days) were unknown.

The allocation of the guinea pigs to the product groups is given inTable 9 in two blocks of six (6) guinea pigs per product group. Noticethat there are two uses of Product Group Numbers 1 and 14. The numbersof the product groups in this table are the same as those identified inTable 1 and were not changed, since the order of these product groupshave been randomized. The person making the observations did not knowwhich product group was used on which animal (blinding).

If the actual experimental variance is less than the estimate used, thenthe study will be more powerful than stated. Alternatively, if theactual experimental variance is more than the estimate used, then thestudy will be less powerful than stated. The design and sample sizecalculations in this memorandum have been guided by information providedby the investigator.

Guinea Pig Model Results and Discussion

I. Effect of Topical Antioxidants on Lesion Viral Replication in GuineaPigs: First Study

The effect of topical antioxidants on lesion virus titers are shown inTable 1. There were no significant differences observed in lesion virustiter-day areas under the curve (AUC) between drug and placebo-treatedanimals.

I. Effect of Topical Antioxidants on Lesion Development in Guinea Pigs:First Study

The effect of topical antioxidants on lesion development are summarizedin Table 2. Only group 9 exhibited a significant reduction in the lesionscore-day AUC when compared to the vehicle treated animals (group 8).Groups 14a, 1a, 7, 18, 8, 10, 1b, 5, 4 (Blistex™), 2, 19, 15, 16 and 20had significantly greater lesion score-day AUC's when compared to theappropriate control group (group 13 or 8).

II. Effect of Topical Antioxidants on Lesion Viral Replication in GuineaPigs: Second Study

The effect of topical antioxidants on lesion virus titers are shown inTable 3. Significant differences were observed in lesion virus titer-dayAUC's in groups 6 (ACV) and 20. It should be noted that group 20 hadonly 4 out of 6 animals with positive vaginal virus titers. Moderatereductions in lesion AUC's were also observed with groups 12 and 2(p-values of 0.06 and 0.0-7, respectively).

IV. Effect of Topical Antioxidants on Lesion Development In Guinea Pigs:Second Study

The effect of topical antioxidants on lesion development are summarizedin Table 4. Groups 11, 17, and 20 exhibited significant reductions inthe lesion score-day AUC when compared to the vehicle treated animals(group 8). Groups 14a, 1a, 18, 9, 5, 4 (Blistex™), 2, 15, 16, 3 and 21had significantly greater lesion score-day AUC's when compared to thevehicle treated animals (group 8). Groups 10 and 1b had moderatelygreater lesion AUC's (p-values of 0.07 and 0.06, respectively).

V. Effect of Topical Antioxidants on Lesion Replication in Guinea Pigs:Combined Results

The effect of topical antioxidants on lesion virus titers from thecombined results of the first and second studies are shown in Table 5.The only significant difference observed in lesion virus titer-day AUC'swas with group 6 (ACV). A moderate reduction in the lesion titer AUC wasshown with group 21 (p-value of 0.07).

VI. Effect of Topical Antioxidants on Lesion Development In Guinea Pigs:Combined Resales

The effect of topical antioxidants on lesion development from thecombined results of the first and second studies are summarized in Table6. Only groups 11 and 17 exhibited a significant reduction in the lesionscore-day AUC when compared to the vehicle treated animals (group 8).Groups 14a, 1a, 18, 8, 9, 10, 1b, 5, 4 (Blistex™), 2, 19, 15, 16, 3 and21 had significantly greater lesion score-day AUC's when compared to theappropriate control animals (groups 13 and 8).

VI. Discussion of Guinea Pig Results

Because of the large number of samples to be tested (22) and the need tocompare directly all of the samples at one time, the study was conductedas two identical experiments with six animals per group. The genitalinfection of guinea pigs is a natural infection and like any biologicalsystem, there is variability from animal to animal in the naturalhistory of the disease and the rate of progression through the variousstages of the primary infection. Due to this variability, a minimumgroup size of 10 guinea pigs per group was established in order tominimize the variability within each group.

In the first second and combined studies, there was excellentcorrelation for effect of treatment on virus titers in lesions. In fact,only the 5% ACV preparation significantly reduced viral replication inexternal genital lesions.

The effect of treatment with the various compounds on the developmentand severity of lesions on the external genital skin was more variablebetween the two experiments, however, almost all of the preparationsresulted in more severe lesions than the untreated control, the vehiclecontrol, or the group treated with ACV. One of the reference compounds(Blistex™, group 4) was consistently worse than the vehicle control.Groups 11 and 17 were the only preparations that significantly reducedthe lesion score compared with the vehicle control. Groups 7, 12, 20 and14b were neutral preparations in that they did not decrease or increasethe severity of the genital lesions. In contrast, groups 18, 10, 1b, 5,4, 2, 15, 16, 3 and 21 clearly resulted in significantly more severedisease than those that were untreated, treated with vehicle alone, orwith ACV-PEG. Analysis of the various components in each of theformulations identifies those materials that contribute to healing orexacerbation of disease severity and the information obtained will beused to construct a more optimal formulation.

Mouse Model Results and Discussion

All groups contained at least 8 animals by the conclusion of the study,however, as many as 16 animals at minimum, were infected for eachtreatment. Mice that did not show clinical signs for at least twoconsecutive days following inoculation of the virus, were considered tobe uninfected and were excluded from the study (the Acyclovir controlwas an exception as this positive control was expected to prevent viralreplication and reduce clinical signs). The infection rate varied from60% to 100% over the course of experiments.

By taking daily measurements of the lesions, a disease curve wasconstructed which consisted of 3 phases of the infection: incubation,log, and resolution. The data, presented as the average lesion area (insq. mm) for the positive (Acyclovir) and negative (PEG) drug treatmentcontrols from a representative experiment, are shown in FIG. 41. Theincubation period for this infection spanned day 0 to day 5 p.i.Measurable herpetic lesions developed in the PEG-treated group between 5and 6 days p.i. The severity of the lesions continued to increase in thelog phase of the infection through day 7 p.i., and peak clinical signsoccurred on day 8 p.i. The resolution phase of the infection occurredfrom day 9 through day 12 p.i. Mice treated with acyclovir showedminimum clinical signs, only 2 out of a total of 18 HSV-infected micedeveloped clinical signs.

In addition to measuring the lesion area on a daily basis, symptomscores from 0 to 4 (see Materials and Methods section) were alsorecorded (FIG. 42). As seen in FIG. 42, this curve tended to have a morebroad pattern. This was due to the fact that infected mice had clinicalsigns of infection, such as erythema and swelling, prior to thedevelopment of the actual herpetic lesions

The "area under the curves" for the lesions area and the clinicalsymptoms was also calculated. These data proved a useful way ofexpressing the dynamics of the infectious disease process. In FIG. 43,the "area under the curve" (y axis) for the clinical symptoms for eachgroup (numbers on the x axis) and the control groups (PEG, Base orBlistex™), represented by the dotted lines, was compared. Data pointsbelow the dotted lines had lower average clinical symptom scores thanthe controls. This analysis showed that compounds 1, 15 and 16 had themost reduced clinical symptoms compared to the control groups.

Summary of Mouse and Guinea Pig Studies

In summary, the guinea pig model produced statistically and clinicallysignificant results, while the mouse model provided clinically but notstatistically significant results. The guinea pig study showed that theconcentration of wound healing components in formulas #11 and #17reduced lesion development, duration, and severity. Acyclovir reducedviral titers, but its effect on viral lesion development, duration, andseverity was worse than groups 11 and 17. In the mouse model, groups 1,15, and 16 reduced clinical symptoms compared to the control group.Acyclovir reduced lesion development, duration, and severity, producingthe best results. The mouse model which is generally used as a screenfor antiviral compounds, was modified in an attempt to broaden itssensitivity to differentiate among the test formula. Unfortunately,lesion size variations increased within each test group, producingnon-statistically significant results for that parameter. In spite ofthese unforeseen problems, some meaningful data was obtained from themouse model experiment. In both models the best formulas contained anequal ratio of Vitamin E and pyruvate and the higher levels of fattyacids. In the guinea pig model, formulas #11 and #17 contained 0.5% ofboth Vitamin E and pyruvate. In the mouse model, formulas #1 and #16contained 4.75% of the both actives therefore suggesting that equal buthigher concentrations of the actives were needed for skin penetration,healing and cell resuscitation. Variation from these ratios reduced thehealing efficacy of the formulas in both models. Both animal studiesconfirmed previous results attained on the efficacy of wound healingcompositions to accelerate wound healing.

FIG. 41 is a graph illustrating the lesion area curves for mice infectedwith herpes simplex virus and treated with acyclovir (ACV, positive) andpolyethylene glycol (PEG, negative). The x-axis represents days postinfection and the y-axis represents the average lesion area (mm²).

FIG. 42 is a graph illustrating the symptom score curves for miceinfected with herpes simplex virus and treated with acyclovir (ACV,positive) and polyethylene glycol (PEG, negative). The x-axis representsdays post infection and the y-axis represents the symptom score.

FIG. 43 is a graph illustrating the area under the symptom score curvesby group for mice infected with herpes simplex virus. The x-axisrepresents the groups and the y-axis represents the area under thesymptom score curve by day 12. The clinical symptoms for each group arerepresented as numbers on the x axis and the control groups(polyethylene glycol, base, or Blistex™) are represented by dottedlines.

FIGS. 44A-44B are photographs illustrating the scoring of cold sorelesions in guinea pig. The scorings illustrated are 1.0 and 1.5. Thescorings range from 0 to 4, with 4 being the worst. FIGS. 45A-45B arephotographs illustrating the scoring of cold sore lesions in guinea pig.The scorings illustrated are 2.0 and 2.5. The scorings range from 0 to4, with 4 being the worst.

FIGS. 46A-46B are photographs illustrating the scoring of cold sorelesions in guinea pig. The scorings illustrated are 3.0 and 3.5. Thescorings range from 0 to 4, with 4 being the worst.

FIGS. 47A-47B are photographs illustrating the scoring of cold sorelesions in guinea pig. The scorings illustrated are 4.0 and 0.0(control). The scorings range from 0 to 4, with 4 being the worst.

FIGS. 48A-48B are photographs illustrating the scoring of cold sorelesions in guinea pig. Animals in FIG.19a were treated with formula 11or 17. Animals in FIG. 19B were treated with BLISTEX™. The scoringsrange from 0 to 4, with 4 being the worst.

FIGS. 49A-49D are photographs illustrating the scoring of cold sorelesions in hairless mice. The scorings illustrated are 1.0, 2.0, 3.0 and4.0, respectively. The scorings range from 0 to 4, with 4 being theworst.

                  TABLE 1    ______________________________________    Evaluation of Topical Antioxidants on Lesion Virus Titers    in a Primary Genital HSV-2 Infection in Guinea Pigs:    First Study                      Lesion             # Vaginal                      Virus           Mean             Virus    Titer-Day       Peak             Positive/                      Area            Lesion             #        Under           Virus    Treatment.sup.a             Inoculated                      Curve    P-Value.sup.b                                      Titer P-Value.sup.b    ______________________________________    Group 13 6/6      16.5     --     3.9   --    (untreated)    Group 11 6/6      18.2     .sup. NS.sup.c                                      4.2   NS    Group 14a             6/6      16.6     NS     3.7   NS    Group 1a 6/6      18.1     NS     3.7   NS    Group 7  6/6      15.9     NS     4.1   NS    Group 12 6/6      17.7     NS     3.9   NS    Group 18 6/6      18.7     NS     4.1   .sup. 0.08.sup.d    Group 6  6/6      9.9      NS     4.0   NS    (5% ACV/    PEG)    Group 8  6/6      19.5     NS     4.5   NS    Group 9  6/6      15.6     NS     3.8   NS    Group 10 6/6      17.0     NS     3.8   NS    Group 1b 6/6      18.4     NS     4.4   NS    Group 5  6/6      18.6     NS     4.1   0.08    Group 4  6/6      16.1     NS     4.0   0.07    (BLISTEX ™)    Group 2  6/6      20.0     NS     4.4   NS    Group 19 6/6      17.4     NS     3.9   0.07    Group 15 6/6      19.1     NS     4.3   NS    Group 16 6/6      19.2     NS     4.3   NS    Group 17 6/6      17.3     NS     4.2   NS    Group 3  6/6      20.0     NS     4.8   NS    Group 20 6/6      19.4     NS     4.4   NS    Group 14b             6/6      19.2     NS     4.4   NS    ______________________________________     .sup.a Treatment was initiated at 48 hours postinoculation. Animals were     treated 4 times per day for 10 days with 0.1 ml applied topically on     external genitalia.     .sup.b All groups were compared with group 8 (vehicle only), except for     group 8 which was compared with the untreated control animals (group 13).     .sup.c Not significantly different from group 8 or group 13.     .sup.d Lesion titer was decreased compared to vehicle treated animals     (group 8).

                  TABLE 2    ______________________________________    Evaluation of Topical Antioxidants on Lesion Severity    in a Primary Genital HSV-2 Infection of Guinea Pigs:    First Study              Lesion Score              Day Area              Area                Mean Peak    Treatment.sup.a              Under Curve                        P-Value.sup.b                                  Lesion Score                                          P-Value.sup.b    ______________________________________    Group 13  23.3      --        2.7     --    (untreated)    Group 11  26.2      NS.sup.c  2.8     NS    Group 14a 38.6      <0.05.sup.d                                  3.9     NS    Group 1a  38.5      <0.01     3.5     NS    Group 7   37.3      <0.01     3.2     NS    Group 12  28.4      NS        3.0     NS    Group 18  46.8      <0.001    3.9     NS    Group 6   31.6      NS        3.2     NS    (5% ACV/PEG)    Group 8   30.5      <0.01.sup.e                                  3.5     NS    Group 9   22.8      <0.05     2.8     NS    Group 10  40.0      <0.01     3.4     NS    Group 1b  45.1      <0.01     4.1     NS    Group 5   40.2      <0.01     3.5     NS    Group 4   37.4      <0.05     3.5     NS    (BLISTEX ™)    Group 2   40.8      <0.01     3.5     NS    Group 19  40.2      <0.01     3.3     <0.05    Group 15  46.4      <0.01     4.3     <0.05    Group 16  42.4      <0.001    3.7     NS    Group 17  25.2      NS        3.2     NS    Group 3   34.7      NS        3.4     NS    Group 20  47.9      <0.01     4.0     NS    Group 14b 25.7      NS        3.0     NS    ______________________________________     .sup.a Treatment was initiated at 48 hours postinoculation. Animals were     treated 4 times per day for 10 days with 0.1 ml applied topically on     external genitalia.     .sup.b All groups were compared with group 8 (vehicle only), except for     group 8 which was compared with the untreated control animals (group 13).     .sup.c Not significantly different from group 8 or group 13.     .sup.d Lesion severity was increased significantly compared to the     appropriate control animals (group 8 or group 13).     .sup.e Lesion severity was decreased significantly compared to vehicle     treated animals (group 8).

                  TABLE 3    ______________________________________    Evaluation of Topical Antioxidants on Lesion Virus Titers    in a Primary Genital HSV-2 Infection in Guinea Pigs:    Second Study                      Lesion             # Vaginal                      Virus           Mean             Virus    Titer-Day       Peak             Positive/                      Area            Lesion             #        Under           Virus    Treatment.sup.a             Inoculated                      Curve    P-Value.sup.b                                      Titer P-Value.sup.b    ______________________________________    Group 13 6/6      9.8      --     2.7   --    (untreated)    Group 11 6/6      10.6     .sup. NS.sup.c                                      2.7   NS    Group 14a             6/6      13.4     NS     3.6   NS    Group 1a 6/6      9.5      NS     3.0   NS    Group 7  6/6      10.3     NS     2.9   NS    Group 12 6/6      8.6      0.06.sup.d                                      3.1   NS    Group 18 6/6      13.8     <0.001 3.3   0.07    Group 6  6/6      2.3      NS     1.6   0.06.sup.e    (5% ACV/PEG)    Group 8  6/6      11.9     NS     3.6   NS    Group 9  6/6      9.8      NS     2.8   NS    Group 10 6/6      10.9     NS     2.8   NS    Group 1b 6/6      11.1     NS     3.0   NS    Group 5  6/6      12.8     NS     3.3   NS    Group 4  6/6      9.5      0.07   2.9   NS    (BLISTEX ™)    Group 2  6/6      8.9      NS     2.6   <0.05    Group 19 6/6      11.6     NS     3.2   NS    Group 15 6/6      13.1     NS     3.6   NS    Group 16 6/6      11.9     NS     2.7   0.07    Group 17 6/6      9.1      NS     2.7   0.07    Group 3  6/6      9.4      NS     2.3   <0.05    Group 20 6/6      5.7      <0.01  2.2   NS    Group 14b             6/6      12.7     NS     3.2   NS    Group 21 12/12    10.5     NS     3.0   NS    ______________________________________     .sup.a Treatment was initiated at 48 hours postinoculation. Animals were     treated 4 times per day for 10 days with 0.1 mls applied topically on     external genitalia.     .sup.b All groups were compared with group 8 (vehicle only), except for     group 8 which was compared with the untreated control animals (group 13).     .sup.c Not significantly different from group 8 or group 13.     .sup.d Lesion titer was decreased compared to vehicle treated animals     (group 8).     .sup.e Lesion titer was increased compared to untreated control animals     (group 13).

                  TABLE 4    ______________________________________    Evaluation of Topical Antioxidants on Lesion Severity    in a Primary Genital HSV-2 Infection of Guinea Pigs:    Second Study              Lesion Score              Day Area              Area                Mean Peak    Treatment.sup.a              Under Curve                        P-Value.sup.b                                  Lesion Score                                          P-Value.sup.b    ______________________________________    Group 13  21.7      --        2.4     --    (untreated)    Group 11  16.5      <0.05.sup.c                                  2.0     0.08    Group 14a 28.7      <0.05.sup.d                                  3.0     .sup. NS.sup.e    Group 1a  38.6      NS        3.8     <0.05    Group 7   24.5      NS        2.5     NS    Group 12  28.8      NS        2.7     NS    Group 18  35.0      <0.01     3.0     NS    Group 6   24.0      NS        2.2     NS    (5% ACV/PEG)    Group 8   25.0      NS        2.8     NS    Group 9   41.6      <0.001    3.5     NS    Group 10  29.1      <0.07     3.0     NS    Group 1b  28.5      <0.06     2.7     NS    Group 5   37.8      0.001     3.3     NS    Group 4   39.5      0.001     3.8     NS    (BLISTEX ™)    Group 2   37.9      <0.001    3.4     NS    Group 19  27.0      NS        2.4     NS    Group 15  50.7      <0.001    4.3     0.01    Group 16  32.7      <0.01     3.1     NS    Group 17  10.3      <0.001    1.7     <0.01    Group 3   36.5      0.001     3.3     NS    Group 20  11.6      <0.001    1.5     0.06    Group 14b 28.5      NS        2.8     NS    Group 21  35.6      <0.01     3.2     NS    ______________________________________     .sup.a Treatment was initiated at 48 hours postinoculation. Animals were     treated 4 times per day for 10 days with 0.1 ml applied topically on     genitalia.     .sup.b All groups were compared with group 8 (vehicle only), except for     group 8 which was compared with the untreated control animals (group 13).     .sup.c Lesion severity was decreased significantly compared to vehicle     animals (group 8).     .sup.d Lesion severity was increased significantly compared to vehicle     animals (group 8).     .sup.e Not significantly different from group 8 or group 13.

                  TABLE 5    ______________________________________    Evaluation of Topical Antioxidants on Lesion Virus Titers    in a Primary Genital HSV-2 Infection in Guinea Pigs:    Combination of Results From the First and Second Studies                      Lesion             # Vaginal                      Virus           Mean             Virus    Titer-Day       Peak             Positive/                      Area            Lesion             #        Under           Virus    Treatment.sup.a             Inoculated                      Curve    P-Value.sup.b                                      Titer P-Value.sup.b    ______________________________________    Group 13 12/12    13.1     --     3.3   --    (untreated)    Group 11 12/12    14.4     .sup. NS.sup.c                                      3.5   NS    Group 14a             12/12    15.0     NS     3.7   NS    Group 1a 12/12    13.8     NS     3.3   0.07    Group 7  12/12    13.1     NS     3.5   NS    Group 12 12/12    13.1     NS     3.5   NS    Group 18 12/12    16.3     NS     3.7   NS    Group 6  12/12    6.1      <0.01.sup.d                                      2.8   0.05    (5% ACV/PEG)    Group 8  12/12    15.7     NS     4.1   <0.05.sup.e    Group 9  12/12    12.7     NS     3.3   0.05    Group 10 12/12    14.0     NS     3.3   NS    Group 1b 12/12    14.7     NS     3.7   NS    Group 5  12/12    15.7     NS     3.7   NS    Group 4  12/12    12.8     NS     3.5   0.07    (BLISTEX ™)    Group 2  12/12    14.5     NS     3.5   NS    Group 19 12/12    14.5     NS     3.5   NS    Group 15 12/12    16.1     NS     4.0   NS    Group 16 12/12    15.6     NS     3.5   NS    Group 17 12/12    13.2     NS     3.4   NS    Group 3  12/12    14.7     NS     3.6   NS    Group 20 12/12    12.6     NS     3.3   NS    Group 14b             12/12    15.8     NS     3.7   NS    Group 21 12/12    10.5     0.07   3.0   <0.01    ______________________________________     .sup.a Treatment was initiated at 48 hours postinoculation. Animals were     treated 4 times per day for 10 days with 0.1 ml applied topically on     external genitalia.     .sup.b All groups were compared with group 8 (vehicle only), except for     group 8 which was compared with the untreated control animals (group 13).     .sup.c Not significantly different from group 8 or group 13.     .sup.d Lesion titer was decreased compared to vehicle treated animals     (group 8).     .sup.e Lesion titer was increased compared to untreated control animals     (group 13).

                  TABLE 6    ______________________________________    Evaluation of Topical Antioxidants on Lesion Severity    in a Primary Genital HSV-2 Infection of Guinea Pigs:    Combination of Results From the First and Second Studies              Lesion Score              Day Area              Area                Mean Peak    Treatment.sup.a              Under Curve                        P-Value.sup.b                                  Lesion Score                                          P-Value.sup.b    ______________________________________    Group 13  22.5      --        2.5     --    (untreated)    Group 11  21.1      <0.05.sup.c                                  2.4     0.07    Group 14a 33.4      <0.05.sup.d                                  3.5     .sup. NS.sup.e    Group 1a  38.5      <0.01     3.5     NS    Group 7   30.5      NS        2.8     NS    Group 12  28.6      NS        2.8     NS    Group 18  40.9      <0.001    3.5     NS    Group 6   27.8      NS        2.7     NS    (5% ACV/PEG)    Group 8   27.4      <0.05     3.1     NS    Group 9   32.7      <0.05     3.2     NS    Group 10  34.9      <0.01     3.2     NS    Group 1b  37.4      <0.01     3.4     NS    Group 5   39.0      0.001     3.4     NS    Group 4   38.6      <0.01     3.7     NS    (BLISTEX ™)    Group 2   39.4      <0.001    3.5     NS    Group 19  32.9      <0.05     2.9     <0.001    Group 15  48.6      <0.001    4.3     NS    Group 16  36.7      <0.01     3.4     0.06    Group 17  17.6      <0.01     2.4     NS    Group 3   35.6      <0.01     3.4     NS    Group 20  28.8      NS        2.8     NS    Group 14b 27.3      NS        2.9     NS    Group 21  35.6      <0.01     3.2     NS    ______________________________________     .sup.a Treatment was initiated at 48 hours postinoculation. Animals were     treated 4 times per day for 10 days with 0.1 ml applied topically on     external genitalia.     .sup.b All groups were compared with group 8 (vehicle only), except for     group 8 which was compared with the untreated control animals (group 13).     .sup.c Not significantly different from group 8 or group 13.     .sup.d Lesion severity was increased significantly compared to the     appropriate control animals (group 8 or group 13).     .sup.e Lesion severity was decreased significantly compared to vehicle     treated animals (group 8).

                  TABLE 7    ______________________________________    Lesion Scoring System for a    Primary Genital HSV Infection in Guinea Pigs    Score         Description    ______________________________________    0.0  Nothing, normal genital skin    0.5  Distinct erythema    1.0  1-2 lesions    1.5  3-5 lesions    2.0  More than 5 lesions    2.5  More than 5 lesions with some coalesced    3.0  Half of the area covered with coalesced lesions    3.5  Greater than half the area covered with coalesced lesions    4.0  As above with some ulceration (less than half the area ulcerated)    4.5  Half of the area ulcerated    5.0  Greater than half the area ulcerated    4.5  As above but with less than half the area crusted (scabbed over)    4.0  Greater than half of the area crusted/scabbed    3.5  As above but with loss of crust on less than half the area    3.0  Loss of crust on half to 3/4 of the area    2.5  Some distinct (larger patches) crust still left    2.0  Less than above but greater than 5 small areas of crust    1.5  3-5 small areas of crust left    1.0  1-2 small areas of crust left    0.5  Distinct erythema    0.0  Nothing, normal genital skin    ______________________________________

                  TABLE 8    ______________________________________    Cold Sore Product Groups in Mice and Guinea Pigs    Amount in Product (%)    Product         Unsaturated                              Sodium    Group Vitamin E Fatty Acids                              Pyruvate                                     Phenol Lidocaine    ______________________________________     1    4.75      4.75      4.75   0.5    0.5     2    9.00      9.00      9.00   0.5    0.5     3    0.50      0.50      9.00   0.5    0.5     4                        Blistex ™     5    9.00      0.50      0.50   0.5    0.5     6                        Acyclovir     7    4.7       0.00      4.75   0.5    0.5     8*   0.00      0.00      0.00   0.0    0.0     9    0.50      9.00      9.00   0.5    0.5    10    4.75      4.75      0.00   0.5    0.5    11    0.50      0.50      0.50   0.5    0.5    12    9.92      4.75      4 75   0.5    0.5    13                        Untreated    14    0.00      0.00      0.00   0.5    0.5    15    4.75      4.75      9.92   0.5    0.5    16    4.75      9.92      4.75   0.5    0 5    17    0.50      9.00      0.50   0.5    0.5    18    9.00      9.00      0.50   0.5    0.5    19    9.00      0.50      9.00   0.5    0.5    20    0.00      4.75      4.75   0.5    0.5    ______________________________________     *base

                  TABLE 9    ______________________________________    Cold Sore Product Guinea Pig Allocation    Block   Product Group Guinea Pig Number    ______________________________________    1       13            1-6            11             7-12            14            13-18            1             19-24            7             25-30            12            31-36            18            37-42            6             43-48            8             49-54            9             55-60            10            61-66            1             67-72            5             73-78            4             79-84            2             85-90            19            91-96            15             97-102            16            103-108            17            109-114            3             115-120            20            121-126            14            127-132    2       13            133-138            11            139-144            14            145-150            1             151-156            7             157-162            12            163-168            18            169-174            6             175-180            8             181-186            9             187-192            10            193-198            1             199-204            5             205-210            4             211-216            2             217-222            19            223-228            15            229-234            16            235-240            17            241-246            3             247-252            20            253-258            14            259-264    ______________________________________

Statistical Analysis

The product (treatment) groups necessary to evaluate the optimalcombination of the components of wound healing (vitamin E, unsaturatedfatty acids, and sodium pyruvate) in the presence of phenol andlidocaine both at 0.5% by weight are incorporated in the experimentaldesign. The design is a two-cubed factorial with six star points and onecenter point with the "control groups" Blistex™, Acyclovir, anduntreated. The ranges of the three components in wound healing are allfrom 0.5% to 9.0% by weight. The product groups are listed in the randomorder presented to the investigator by number without any otheridentification.

The measures of efficacy considered for statistical analyses are thearea under the lesion score "curve", the peak lesion score, the time toresolution of the lesions, the area under the viral titer "curve", andthe peak viral titer. For each of these measures, the followingstatistical procedure was used. The treatment groups involving woundhealing were all compared to the treatment groups Blistex™, Acyclovir,and base (Treatment Groups 4, 6, and 14, respectively). The factorialportion of the treatment groups were then investigated to determine aresponse model for the components of wound healing. The model was thenused to predict what response could be expected for combinations of thewound healing components that were not statistically different from theminimum response.

Statistical Analysis of the Guinea Pig Model

The guinea pigs were allocated to the product groups in a random orderto two blocks of six (6) guinea pigs per product group with repeats ofProduct Group Numbers 1 and 14 (design center point and base,respectively). The lesion scores for guinea pigs were recorded daily ondays 3 through 19 on a scale of 0 to 5 in half-unit increments inincreasing severity, while the titers were recorded on days 3, 4, 5, 6,7, and 10 as actually measured on a continuous scale. The areas underthe lesion and titer "curves" were calculated with the application ofthe trapezoidal rule. Any missing values were considered as zero valueson both scales. The peaks are the maximum values over the days observed.The time to resolution (healing time) of the lesions was defined asmidway between the last day of non-zero responses and zero responses. Ifno resolution occurred in the time frame of the study, the time toresolution was the last day plus a half day.

For area under the lesion score "curve", Treatment Groups 11 and 17 werestatistically different from Blistex™ (4) (p values of 0.0160 and0.0034, respectively), Treatment Group 17 was almost statisticallydifferent from base (14) (p value of 0.0502), and no treatment groupswere statistically different from Acyclovir (6). For peak lesion score,Treatment Groups 6, 7, 11, 12, 17, and 20 were statistically differentfrom Blistex™ (p values of 0.0178, 0.0479, 0.0031, 0.0479, 0.0031, and0.0297, respectively), Treatment Groups 11 and 17 were statisticallydifferent from base (p values of 0.0347 and 0.0347, respectively), andno treatment groups were statistically different from Acyclovir, exceptBlistex™. For time to lesion resolution, Treatment Groups 11, 17, and 20were statistically different from Blistex™ (p values of 0.0185, 0.0099,and 0.0283, respectively), Treatment Groups 11, 17, and 20 werestatistically different from base (p values of 0.0001, 0093, and 0.0312,respectively), and no treatment groups were statistically different fromAcyclovir. For area under the viral titer "curve", no treatment groupswere statistically different from Blistex™ and base, except Acyclovir,and all treatment groups were statistically different from Acyclovir.For peak viral titer, no treatment groups were statistically differentfrom Blistex™ and base, and Treatment Groups 1, 3, 5, 8, 12, 14, 15, 18,19, and 21 were statistically different from Acyclovir (p values of0.0195, 0.0342, 0.0128, 0.0005, 0.0479, 0.0035, 0.0009, 0.0098, 0.0383,and 0.0174, respectively).

Statistical Analysis of the Mouse Model

The mice were planned to be allocated to the treatment (product) groupsin a random order to one block of eight (8) mice per product group withrepeats of Product Group Numbers 1 and 14 (design center point and base,respectively). The lesion scores for mice were recorded daily on days 1through 14 on a scale of 0 to 4 in half-unit increments in increasingseverity and the lesion areas were measured on a continuous scale onthese same days. The areas under the lesion area and score "curves" werecalculated with the application of the trapezoidal rule. Any missingvalues were considered as zero on both scales. The peaks are the maximumvalues over the days observed. The time to resolution of the lesions wasdefined as midway between the last day of non-zero lesion scores andzero lesion scores. If no resolution occurred in the time frame of thestudy, the time to resolution was the last day plus a half day. Theactual allocation of the mice to the treatment groups was accomplishedon four (4) exposure dates. This resulted in an unbalanced design acrossexposure days. The means presented in this report are adjusted for thisunbalance, probably resulting in an increase in expected variation ofthe measures.

For area under the lesion area "curve", no treatment groups werestatistically different from Blistex™ and base, except Acyclovir, andall treatment groups except Treatment Groups 5 and 15 were statisticallydifferent from Acyclovir. For peak lesion area, no treatment groups werestatistically different from Blistex™ and base, except Acyclovir, andall treatment groups except Treatment Groups 17 and 19 werestatistically different from Acyclovir. For area under the lesion score"curve", no treatment groups were statistically different from Blistex™and base, except Acyclovir, and all treatment groups were statisticallydifferent from Acyclovir. For peak lesion score, no treatment groupswere statistically different from Blistex™ and base, except Acyclovir,and all treatment groups were statistically different from Acyclovir.For time to resolution of lesion scores, no treatment groups werestatistically different from Blistex™ and base, and all treatment groupswere statistically different from Acyclovir (6).

Conclusions

Various combinations of the components of wound healing in theneighborhood of 9% unsaturated fatty acids, 0.5% sodium pyruvate, and0.5% vitamin E statistically reduced the severity, intensity, andduration of coldsore lesions in the guinea pig model in comparison toBlistex™, Acyclovir, and base. Only Acyclovir statistically reducesthese measures of efficacy in the mouse model.

Summary Analysis of the Data from Study 1

Six to eight week old male SKH-1 hairless mice (Charles River) wereinfected with HSV-1 virus, McIntyre strain. Infection was achieved undergeneral anesthesia (Ketamine, Xylazine) by the abrasion of a 1 cm squarearea (using a 25 gauge needle) centrally located on the dorsal surfaceof a mouse. Virus was then applied directly onto the abraded area (10 μlof a 1×10⁹ PFU/ml virus stock). Following inoculation of 10×10⁶ PFU ofHSV-1 McIntyre strain by scarification of the epidermis, herpeticlesions developed by day 5 and persisted through day 12 post infection(p.i.). The viral lesions spread in a zosteriform pattern from the siteof inoculation (middle of the back) to the abdominal area. By day 10,lesions were crested over and complete healing generally occurred by day12 pi.

Individual mice were treated with test compounds starting on theafternoon of the infection day, and treatment was continued for 14 days.Treatments were administered at 7 a.m. and 4 p.m. each day and involvedthe use of a sterile cotton tipped applicator in such a manner that theaffected area was evenly coated with the test compound. If no lesionswere visible, only the site of infection was treated. Data includinglesion scores, number of lesions, and lesion areas were recorded duringthe 7 a.m. treatment session. Each animal was recorded as having one offive possible scores: 0, no signs; 1, redness and swelling; 2, singlelesion; 3, multiple lesions; 4, a spread of lesions in a dermatonepattern. In addition, the actual lesion area on the skin was measuredusing a micrometer (x and y axial values for each lesion were obtainedin millimeters and then multiplied together to give the lesion area).For analysis, the individual lesion scores or areas within a treatmentgroup were averaged on a daily basis. The scores are set out in theTable below.

    ______________________________________    Table of Results (I.A-D + M2)                       3        4                       Mean     Sum of                                      5               2       Lesion   Lesion                                      Mean    1          Lesion  Peak Area                                Peaks Peak   6    Treatment  size    Severity 0-4   Averages                                             Days to    Groups     (mm.sup.2)                       (mm.sup.2)                                Scale Severity                                             Healing    ______________________________________    1 - Control    130.8   51.1   17.5  3.38   12    2 - Pyruvate & 110.4   40.9   18.5  3.14   12        Vitamin E    3 - Vitamin E &                   100.2   36.9   16.8  3.43   11.8        Fatty Acids    4 - Pyruvate & 128.7   48.3   18.1  3.24   12        Fatty Acids    5 - Pyruvate & 87.8    32.2   15.2  3.01   11.9        Vitamin E &        Fatty Acids        (wound healing        composition)    ______________________________________

Inspection of the results clearly shows that the three component woundhealing composition treatment achieved the best results in four of thefive measures (lesion size, lesion severity, sum of lesion peaks, andlesion peak severity).

The following proposed model was established to determine unexpectedresults from the wound healing composition.

    Change from control=Vitamin E+Fatty Acids+Sodium Pyruvate+Synergy

where Vitamin E represents the single component contribution of VitaminE, Fatty Acids is the single component effect of Fatty Acids, SodiumPyruvate is the single component effect of Sodium Pyruvate, and Synergyis the unexplained wound healing composition synergy effect of the threecomponents together.

Applying the data set out above in column 2 of the Table of Results(I.A-D+M) in this model provides the following results.

Lesion Size From (Column 2) in the Table of Results.

Vitamin E+Sodium Pyruvate=110.4-130.8 mm²

Vitamin E+Fatty Acids=100.2-130.8 mm²

Fatty Acids+Sodium Pyruvate=128.7-130.8 mm²

Vitamin E+Fatty Acids+Sodium Pyruvate+Synergy=87.8-130.8 mm²

This model now provides a linear equation system with four equations andfour unknowns, which can be solved by standard mathematical techniquesto give:

Vitamin E=-24.5

Sodium Pyruvate=+4

Fatty Acids=-6.1

Synergy=-16.4

Solving for the remaining columns of the Table of Results (I.A-D+M) inthis model provides the following results.

    ______________________________________    Table of Results of Contribution of Each Component (I.A-D + M2)                       3        4                       Mean     Sum of                                      5               2       Lesion   Lesion                                      Mean    1          Lesion  Peak Area                                Peaks Peak   6    Individual size    Severity 0-4   Averages                                             Days to    Effects    (mm.sup.2)                       (mm.sup.2)                                Scale Severity                                             Healing    ______________________________________    1 - Vitamin E  -24.5   -10.8  -0.15 0.0    -0.1    2 - Pyruvate   +4.0    +0.6   +1.15 -0.2   +0.1    3 - Fatty Acids                   -6.1    -3.4   -0.55 0.0    -0.1    5 - Pyruvate & -16.4   -5.3   -2.75 -0.2   0.0        Vitamin E &        Fatty Acids        (wound healing        composition)    ______________________________________

Comparing the predicted effect of the wound healing composition with theactual effect of wound healing composition gives the difference ineffect of wound healing composition and the % synergy difference set outbelow.

    ______________________________________    Table of Results    Showing Contribution of Wound Healing Composition (I.A-D + M2)                     3         4      5             2       Mean Lesion                               Sum of Mean             Lesion  Peak Area Lesion Peak   6             Size    Severity  Peaks 0-4                                      Averages                                             Days to    1        (mm.sup.2)                     (mm.sup.2)                               Scale  Severity                                             Healing    ______________________________________    Predicted             -26.6   -13.6     +0.45  -0.2   0    Effect of    Wound Healing    Composition    Actual Effect             -43.0   -18.9     -2.3   -0.4   +0.1    of Wound    Healing    Composition    Difference in             -16.4    -5.3     -2.75  -0.2   +0.1    Effect of    Wound Healing    Composition    % Synergy             61%     39%       611%   100%   0    Difference    ______________________________________

As shown in the Table of Results Showing Contribution of Wound HealingComposition (I.A-D+M2), the wound healing composition reduced four ofthe five lesion measurements greater than was predicted for the threecomponent effect. The wound healing composition reduced lesion sizes 61%more than was predicted for the three component effect. The woundhealing composition reduced mean lesion peak are (severity) 39% greaterthan was predicted for the three component effect. The wound healingcomposition reduced the sum of lesion peaks 611% greater than waspredicted for the three component effect. The wound healing compositionalso reduced mean peak averages 100% greater than was predicted for thethree component effect. The wound healing composition did not reduce thedays to healing in this model because mice require approximately fivedays to develop lesions, three days to respond (inflammatory phase),leaving only four days to heal. The effect of the wound healingcomposition to reduce is small in this model because the wound healingcomposition is not an antiviral agent and can only enhance healing afterthe inflammatory phase is over. In these examples, the antiviral agentwas phenol, which does not effectively reduce viral titers, and lesionsdo not heal until the viral infection is reduced. In summary, the woundhealing composition provided greater results than was predicted for thethree component. The wound healing composition was synergistic inreducing viral lesions and severity but did not reduce times to healingin this model.

Examples of the Antiviral-Wound Healing Compositions of Embodiment Two(I.A-D+M2) Study 2

This study is a summary of findings from an evaluation of a cold soretreatment product conducted among 20 patients who presented with theonset of symptoms of herpes labialus. The results from this study areillustrated in examples 22-41 below.

Twenty subjects were enrolled and completed the expert study. Eachsubject was administered a lip balm containing the wound healingcomposition, 2% sodium pyruvate, 2% vitamin E acetate, and 4% chickenfat, in a Lubriderm lotion base. Enrollment was based on the presence ofvesiculation and a positive viral assay for herpes. Treatment with thestudy product began immediately following the positive viral assay andcontinued for up to two (2) weeks depending upon when full resolution,i.e, elimination of scabbing, occurred.

Table 1 (not shown) is a summary of daily diary responses which showsthat pain, itching, and swelling predominated over the first three (3)days (Baseline, Day 2 and Day 3) after vesicle formation had occurredand thereafter diminished. Pain was no longer present in any subject byDay 7 and in only one (1) subject by Day 5. Itching extended slightlyfurther with four (4) subjects still reporting mild itching by Day 6,two (2) subjects by Days 7 and 8, one (1) subject by Day 9 and nosubjects by Day 10. The pattern for swelling closely paralleled that forpain with only one (1) subject reporting mild swelling on Days 6 and 7.

Results of clinical examinations are summarized in Table 2 (not shown).It is important to note in examining these tables that apparentday-to-day fluctuations in data are due to 1) the every-other-day visitschedule and 2) the fact that subjects were not on the same visitschedule. (Depending upon the day of the week on which subjects wereenrolled, their return visits would be adjusted to accommodate theweekends). Thus, data can be analyzed only for general trends.

Results show that vesiculation persisted in approximately two-thirds(2/3) of the patients to Day 3 and thereafter diminished significantly.There was no evidence of ulceration occurring with any subject at anyinterval. Scabbing was present in more than half of the subjects by Day3 and persisted at a relatively high level through Day 8 after which itdiminished significantly. Full resolution was noted for seven (7)subjects on Day 8 with an additional six (6) subjects on Day 9 (twosubjects), Day 10 (three subjects) and Day 11 (one subject). Theremaining seven (7) subjects were found to be fully resolved on Day 13(four subjects), Day 14 (two subjects) and Day 15 (one subject). Whenasked at their examination visits to assess pain, six (6) subjectsreported mild pain on Day 3 with no pain reported by any subject beyondthat interval. The severity of pruritis diminished significantly fromBaseline (5 moderate and 2 severe) to Day 3 (1 moderate and no severe)with a few subjects reporting mild pruritis between Days 4 and 8.

Table 3 (not shown) is a summary of the comparison of resolution time asdetermined by clinical examination and by the patient's recollection ofhistorical resolution time from prior incidence. No advantage in time tocold sore resolution is evident from examination of this data. Thenumber of days required for scabbing to occur was also clinicallyobserved and, in those instances in which the patient defined resolutionas scab formation, there still appeared to be no advantage to the studyproduct.

Subjective responses to questionnaires are summarized in Table 4 (notshown). Thirteen (13) of 20 subjects regarded the product as excellent,six (6) as good and one (1) poor. Eleven of the 13 subjects who ratedthe product as excellent claimed that it worked fast or faster thanother medications. Fourteen of the study participants regarded the coldsore as not as bad as normal, five (5) regarded it as about the same andone (1) as worse than normal. Regarding relief of pain, discomfortand/or soreness, 12 of 20 subjects regarded it as excellent and eight(8) as good.

In conclusion, under the conditions employed in this study, there wereno apparent increases in healing rate as the result of the use of thestudy product in the treatment of cold sores. However, the product wasperceived by approximately two-thirds (2/3) of the patients to beexcellent and to work fast or faster than other medications. Inaddition, the majority of subjects perceived the cold sore to be not asbad as normal and rated the product as excellent for the relief of pain,discomfort and/or soreness. Finally only one (1) adverse event occurred,namely, one (1) subject who developed an additional cold sore lesion.However, the development of the additional lesion is not regarded asproduct-related.

3. The Antikeratolytic-Wound Healing Compositions a. Embodiment Two(I.A-D+M3)

Applicant has discovered therapeutic antikeratolytic-wound healingcompositions (I.A-D+M3) which comprise an antikeratolytic agent (M3) andthe wound healing compositions of Embodiment One (I.A-D). Preferably,the wound healing composition (I.A) comprises (a) pyruvate, (b) anantioxidant, and (c) a mixture of saturated and unsaturated fatty acids.Antikeratolytic agents can reduce scaling and dryness in a patient butdo not promote the wound healing process. Wound healing compositions canincrease the resuscitation rate of injured mammalian cells and theproliferation rate of new mammalian cells to replace dead cells but donot reduce the proliferation of cells. Applicants have found that thecombination of an antikeratolytic agent and a wound healing compositionresults in a therapeutic antikeratolytic-wound healing composition whichreduces the duration and severity of psoriasis.

The combination of the antikeratolytic agent and the wound healingcompositions of the present invention provides a pharmaceuticalcomposition useful for reducing scaling and dryness and having anenhanced ability to prevent and reduce injury to mammalian cells andfurther increase the resuscitation rate of injured mammalian cells. Thetissue damage associated with psoriasis is believed to be caused by theproduction of cellular produced active oxygen species. Combination ofthe antikeratolytic agent and the wound healing compositions maysuppress such reactive oxygen-linked tissue injury.

The antikeratolytic agents in the antikeratolytic-wound healingcompositions of the present invention may be selected from a widevariety of water-soluble and water-insoluble drugs and their acidaddition or metallic salts. Both organic and inorganic salts may be usedprovided the antikeratolytic agent maintains its medicament value. Theantikeratolytic agents may be selected from a wide range of therapeuticagents and mixtures of therapeutic agents which may be administered insustained release or prolonged action form. Nonlimiting illustrativecategories and specific examples of such antikeratolytic agents includethe following medicaments.

(a) Salicylic acid (2-hydroxybenzoic acid) is an antikeratolytic agentwhich mechanically removes epidermal cells, softens the stratum corneum,and promotes hydration;

(b) Lactic acid is a naturally occurring humectant in the skin whichslows the proliferation of cells and has beneficial effects on dry skinand on severe hyperantikeratolytic conditions; and

(c) Urea is an effective antikeratolytic agent which slows theproliferation of cells.

Preferred antikeratolytic agents to be employed may be selected from thegroup consisting of salicylic acid, lactic acid, and urea. In apreferred embodiment, the antikeratolytic agent is selected from thegroup consisting of salicylic acid and lactic acid. In a more preferredembodiment, the antikeratolytic agent is salicylic acid.

The antikeratolytic agent of the present invention may be used in manydistinct physical forms well known in the pharmaceutical art to providean initial dosage of the antikeratolytic agent and/or a furthertime-release form of the antikeratolytic agent. Without being limitedthereto, such physical forms include free forms and encapsulated forms,and mixtures thereof.

The amount of antikeratolytic agent used in the present invention mayvary depending upon the therapeutic dosage recommended or permitted forthe particular antikeratolytic agent. In general, the amount ofantikeratolytic agent present is the ordinary dosage required to obtainthe desired result. Such dosages are known to the skilled practitionerin the medical arts and are not a part of the present invention. In apreferred embodiment, the antikeratolytic agent in theantikeratolytic-wound healing composition is present in an amount fromabout 0.1% to about 12%, preferably from about 1% to about 10%, and morepreferably from about 2% to about 7%, by weight.

In yet another preferred embodiment, the therapeuticantikeratolytic-wound healing compositions of the present inventionfurther comprise a topical anesthetic agent such as pramoxinehydrochloride, lidocaine, or benzocaine. The amount of anesthetic agentused in the present invention may vary depending upon the therapeuticdosage recommended or permitted for the particular anesthetic agent. Ingeneral, the amount of anesthetic agent present is the ordinary dosagerequired to obtain the desired result. Such dosages are known to theskilled practitioner in the medical arts and are not a part of thepresent invention.

b. Methods For Making the Antikeratolytic-Wound Healing Compositions ofEmbodiment Two (I.A-D+M3)

The present invention extends to methods for making the therapeuticantikeratolytic-wound healing compositions (I.A-D+M3). In general, atherapeutic antikeratolytic-wound healing composition is made by formingan admixture of the wound healing components of Embodiment One (I.A-D)and an antikeratolytic agent. In a first aspect of Embodiment Two(I.A+M3), an antikeratolytic-wound healing therapeutic composition ismade by forming an admixture of an antikeratolytic agent and a woundhealing composition comprising (a) a pyruvate, (b) an antioxidant, and(c) a mixture of saturated and unsaturated fatty acids. In a secondaspect of Embodiment Two (I.B+M3), an antikeratolytic-wound healingtherapeutic composition is made by forming an admixture of anantikeratolytic agent and a wound healing composition comprising (a) apyruvate, (b) a lactate, and (c) a mixture of saturated and unsaturatedfatty acids. In a third aspect of Embodiment Two (I.C+M3), anantikeratolytic-wound healing therapeutic composition is made by formingan admixture of an antikeratolytic agent and a wound healing compositioncomprising (a) an antioxidant, and (b) a mixture of saturated andunsaturated fatty acids. In a fourth aspect of Embodiment Two (I.D+M3),an antikeratolytic-wound healing therapeutic composition is made byforming an admixture of an antikeratolytic agent and a wound healingcomposition comprising (a) a lactate, (b) an antioxidant, and (c) amixture of saturated and unsaturated fatty acids.

In a preferred embodiment, the invention is directed to a method forpreparing a therapeutic antikeratolytic-wound healing composition(I.A+M3) which comprises the steps of admixing the followingingredients:

(A) a therapeutically effective amount of an antikeratolytic agent; and

(B) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

c. Methods For Employing the Antikeratolytic-Wound Healing Compositionsof Embodiment Two (I.A-D+M3)

The present invention extends to methods for employing the therapeuticantikeratolytic-wound healing compositions (I.A-D+M3). In general, atherapeutic composition is employed by contacting the therapeuticcomposition with a wound. In a preferred embodiment, the invention isdirected to a method for healing a psoriasis wound in a mammal with anantikeratolytic-wound healing composition (I.A+M3) which comprises thesteps of:

(A) providing a therapeutic antikeratolytic-wound healing compositionwhich comprises:

(1) a therapeutically effective amount of an antikeratolytic agent; and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) contacting the antikeratolytic-wound healing composition with thepsoriasis wound.

d. Augmented Antikeratolytic-Wound Healing Compositions of EmbodimentTwo (I.A-D+M3+M)

In another aspect of Embodiment Two, the therapeuticantikeratolytic-wound healing compositions (I.A-D+M3) of the presentinvention may be further combined with medicaments useful for treatingwounds (M) to form augmented antikeratolytic-wound healing compositions(I.A-D+M3+M). In this embodiment, the combination of theantikeratolytic-wound healing composition of the present invention andthe medicament useful for treating wounds provides an augmentedantikeratolytic-wound healing composition having an enhanced ability toincrease the proliferation and resuscitation rate of mammalian cells.For example, the therapeutic compositions of the present invention maybe used in combination with medicaments useful for treating wounds suchas immunostimulating agents (Betafectin™), antiviral agents, otherantikeratolytic agents, anti-inflammatory agents, antifungal agents,tretinoin, sunscreen agents, dermatological agents, topicalantihistamine agents, antibacterial agents, bioadhesive agents,respiratory bursting inhibitors (lactic acid, adenosine), inhibitors ofprostaglandin synthesis (ibuprofen, aspirin, indomethacin, meclofenomicacid, retinoic acid, padimate O, meclomen, oxybenzone), steroidalanti-inflammatory agents (corticosteroids including synthetic analogs),antimicrobial agents (neosporin ointment, silvadine), antiseptic agents,anesthetic agents (pramoxine hydrochloride, lidocaine, benzocaine), cellnutrient media, burn relief medications, sun burn medications, acnepreparations, insect bite and sting medications, wound cleansers, wounddressings, scar reducing agents (vitamin E), and the like, and mixturesthereof, to further enhance the proliferation and resuscitation rate ofmammalian cells. Preferably, the medicament useful for treating woundsis selected from the group consisting of immunostimulating agents,antiviral agents, antikeratolytic agents, anti-inflammatory agents,antifungal agents, tretinoin, sunscreen agents, dermatological agents,topical antihistamine agents, antibacterial agents, bioadhesive agents,respiratory bursting inhibitors, inhibitors of prostaglandin synthesis,antimicrobial agents, cell nutrient media, scar reducing agents, andmixtures thereof. More preferably, the medicament useful for treatingwounds is selected from the group consisting of immunostimulatingagents, antiviral agents, antikeratolytic agents, anti-inflammatoryagents, antifungal agents, acne treating agents, sunscreen agents,dermatological agents, antihistamine agents, antibacterial agents,bioadhesive agents, and mixtures thereof.

In a preferred embodiment, the invention is directed to an augmentedantikeratolytic-wound healing composition (I.A+M3+M) which comprises:

(A) a therapeutic antikeratolytic-wound healing composition whichcomprises:

(1) a therapeutically effective amount of an antikeratolytic agent; and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a medicament useful for treating wounds.

The present invention extends to methods for making the augmentedantikeratolytic-wound healing compositions. In general, the augmentedcompositions are made by admixing the therapeutic antikeratolytic-woundhealing composition with the medicament useful for treating wounds toprepare the augmented antikeratolytic-wound healing composition.

The present invention also extends to methods for employing theaugmented antikeratolytic-wound healing compositions. In general, anaugmented antikeratolytic-wound healing composition is employed bycontacting the composition with a wound. In a preferred embodiment, theinvention is directed to a method for healing a psoriasis wound in amammal with an augmented antikeratolytic-wound healing composition(I.A+M3+M) which comprises the steps of:

(A) providing a therapeutic augmented antikeratolytic-wound healingcomposition which comprises: (1) a therapeutically effective amount ofan antikeratolytic agent; (2) a wound healing composition whichcomprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(3) providing a medicament useful for treating wounds; and

(B) contacting the augmented antikeratolytic-wound healing compositionwith the psoriasis wound.

The types of wounds which may be healed using the antikeratolytic-woundhealing compositions and the augmented antikeratolytic-wound healingcompositions of the present invention are those which result from apsoriasis injury which causes epidermal damage.

Methods for healing a wound comprise topically administering thecompositions of the present invention directly to a wound site toincrease the healing rate of the wound. The composition is maintained incontact with the wound for a period of time sufficient to increase theproliferation and resuscitation rate of the cells.

e. Formulations of the Antikeratolytic-Wound Healing Compositions ofEmbodiment Two (I.A-D+M3) and (I.A-D+M3+M)

Once prepared, the inventive therapeutic antikeratolytic-wound healingcompositions and augmented antikeratolytic-wound healing compositionsmay be stored for future use or may be formulated in effective amountswith pharmaceutically acceptable carriers such as pharmaceuticalappliances and topical vehicles (oral and non-oral) to prepare a widevariety of pharmaceutical compositions. The pharmaceutically acceptablecarriers which may be employed and the methods used to prepare thepharmaceutical compositions have been described above in connection withthe formulations of the wound healing compositions of Embodiment One(I.A-D).

In a preferred embodiment, the invention is directed to anantikeratolytic-wound healing pharmaceutical composition whichcomprises:

(A) a therapeutic antikeratolytic-wound healing composition (I.A+M3)which comprises:

(1) a therapeutically effective amount of an antikeratolytic agent; and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a pharmaceutically acceptable carrier selected from the groupconsisting of pharmaceutical appliances, bioadhesives, and occlusivevehicles.

In another preferred embodiment, the invention is directed to a methodfor preparing a pharmaceutical composition for increasing theproliferation and resuscitation rate of mammalian cells, which comprisesthe steps of:

(A) providing a therapeutically effective amount of anantikeratolytic-wound healing composition (I.A+M3) which comprises:

(1) an antikeratolytic agent; and

(2) a wound healing composition comprising:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(B) providing a pharmaceutically acceptable carrier; and

(C) admixing the antikeratolytic-wound healing composition from step (A)and the pharmaceutically acceptable carrier from step (B) to form apharmaceutical composition.

4. Anti-Inflammatory-Wound Healing Compositions a. Embodiment Two(I.A-D+M4)

Applicant has discovered therapeutic anti-inflammatory-wound healingcompositions (I.A-D+M4) which comprise an anti-inflammatory agent (M4)and the wound healing compositions of Embodiment One (I.A-D).Preferably, the wound healing composition (I.A) comprises (a) pyruvate,(b) an antioxidant, and (c) a mixture of saturated and unsaturated fattyacids. Anti-inflammatory agents can reduce inflammation in a patient butdo not promote the wound healing process. Wound healing compositions canincrease the resuscitation rate of injured mammalian cells and theproliferation rate of new mammalian cells to replace dead cells but donot reduce inflammation. Applicants have found that the combination ofan anti-inflammatory agent and a wound healing composition results in atherapeutic anti-inflammatory-wound healing composition which reducesthe duration and severity of inflammation.

The combination of the anti-inflammatory agent and the wound healingcompositions of the present invention provides a pharmaceuticalcomposition useful for reducing inflammation and having an enhancedability to prevent and reduce injury to mammalian cells and furtherincrease the resuscitation rate of injured mammalian cells. The tissuedamage associated with inflammation is believed to be caused by theproduction of cellular produced active oxygen species. Combination ofthe anti-inflammatory agent and the wound healing compositions maysuppress such reactive oxygen-linked tissue injury.

The anti-inflammatory agents in the anti-inflammatory-wound healingcompositions of the present invention may be selected from a widevariety of steroidal, non-steroidal, and salicylate water-soluble andwater-insoluble drugs and their acid addition or metallic salts. Bothorganic and inorganic salts may be used provided the anti-inflammatoryagent maintains its medicament value. The anti-inflammatory agents maybe selected from a wide range of therapeutic agents and mixtures oftherapeutic agents which may be administered in sustained release orprolonged action form. Nonlimiting illustrative specific examples ofnon-steroidal anti-inflammatory agents include the followingmedicaments: ibuprofen, naproxen, sulindac, diflunisal, piroxicam,indomethacin, etodolac, meclofenamate sodium, fenoproben calcium,ketoprofen, mefenamic acid, nabumetone, ketorolac tromethamine,diclofenac, and evening primrose oil (containing about 72% linoleic acidand about 9% gamma-linolenic acid). Nonlimiting illustrative specificexamples of salicylate anti-inflammatory agents include the followingmedicaments: acetylsalicylic acid, mesalamine, salsalate, diflunisal,salicylsalicylic acid, and choline magnesium trisalicylate. Nonlimitingillustrative specific examples of steroidal anti-inflammatory agentsinclude the following medicaments: flunisolide, triamcinoline,triamcinoline acetonide, beclomethasone diproprionate, betamethasonediproprionate, hydrocortisone, cortisone, dexamethasone, prednisone,methyl prednisolone, and prednisolone.

Preferred anti-inflammatory agents to be employed may be selected fromthe group consisting of ibuprofen, naproxen, sulindac, diflunisal,piroxicam, indomethacin, etodolac, meclofenamate sodium, fenoprobencalcium, ketoprofen, mefenamic acid, nabumetone, ketorolac tromethamine,diclofenac, evening primrose oil, acetylsalicylic acid, mesalamine,salsalate, diflunisal, salicylsalicylic acid, choline magnesiumtrisalicylate, flunisolide, triamcinoline, triamcinoline acetonide,beclomethasone diproprionate, betamethasone diproprionate,hydrocortisone, cortisone, dexamethasone, prednisone, methylprednisolone, and prednisolone. In a preferred embodiment, theanti-inflammatory agent is selected from the group consisting ofibuprofen, naproxen, sulindac, diflunisal, piroxicam, indomethacin,etodolac, meclofenamate sodium, fenoproben calcium, ketoprofen,mefenamic acid, nabumetone, ketorolac tromethamine, diclofenac, andevening primrose oil. In a more preferred embodiment, theanti-inflammatory agent is evening primrose oil.

The anti-inflammatory agent of the present invention may be used in manydistinct physical forms well known in the pharmaceutical art to providean initial dosage of the anti-inflammatory agent and/or a furthertime-release form of the anti-inflammatory agent. Without being limitedthereto, such physical forms include free forms and encapsulated forms,and mixtures thereof.

The amount of anti-inflammatory agent used in the present invention mayvary depending upon the therapeutic dosage recommended or permitted forthe particular anti-inflammatory agent. In general, the amount ofanti-inflammatory agent present is the ordinary dosage required toobtain the desired result. Such dosages are known to the skilledpractitioner in the medical arts and are not a part of the presentinvention. In a preferred embodiment, the anti-inflammatory agent in theanti-inflammatory-wound healing composition is present in an amount fromabout 0.01% to about 10%, preferably from about 0.1% to about 5%, andmore preferably from about 1% to about 3%, by weight.

In yet another preferred embodiment, the therapeuticanti-inflammatory-wound healing compositions of the present inventionfurther comprise a topical antipruritic agent such as menthol. Theamount of antipruritic agent used in the present invention may varydepending upon the therapeutic dosage recommended or permitted for theparticular anesthetic agent. In general, the amount of antipruriticagent present is the ordinary dosage required to obtain the desiredresult. Such dosages are known to the skilled practitioner in themedical arts and are not a part of the present invention.

In still yet another preferred embodiment, the therapeuticanti-inflammatory-wound healing compositions of the present inventionfurther comprise a decongestant agent such as eucalyptus oil.

b. Methods For Making the Anti-Inflammatory-Wound Healing Compositionsof Embodiment Two (I.A-D+M4)

The present invention extends to methods for making the therapeuticanti-inflammatory-wound healing compositions (I.A-D+M4). In general, atherapeutic anti-inflammatory-wound healing composition is made byforming an admixture of the wound healing components of Embodiment One(I.A-D) and an anti-inflammatory agent. In a first aspect of EmbodimentTwo (I.A+M4), an anti-inflammatory-wound healing therapeutic compositionis made by forming an admixture of an anti-inflammatory agent and awound healing composition comprising (a) a pyruvate, (b) an antioxidant,and (c) a mixture of saturated and unsaturated fatty acids. In a secondaspect of Embodiment Two (I.B+M4), an anti-inflammatory-wound healingtherapeutic composition is made by forming an admixture of ananti-inflammatory agent and a wound healing composition comprising (a) apyruvate, (b) a lactate, and (c) a mixture of saturated and unsaturatedfatty acids. In a third aspect of Embodiment Two (I.C+M4), ananti-inflammatory-wound healing therapeutic composition is made byforming an admixture of an anti-inflammatory agent and a wound healingcomposition comprising (a) an antioxidant, and (b) a mixture ofsaturated and unsaturated fatty acids. In a fourth aspect of EmbodimentTwo (I.D+M4), an anti-inflammatory-wound healing therapeutic compositionis made by forming an admixture of an anti-inflammatory agent and awound healing composition comprising (a) a lactate, (b) an antioxidant,and (c) a mixture of saturated and unsaturated fatty acids.

In a preferred embodiment, the invention is directed to a method forpreparing a therapeutic anti-inflammatory-wound healing composition(I.A+M4) which comprises the steps of admixing the followingingredients:

(A) a therapeutically effective amount of an anti-inflammatory agent;and

(B) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

c. Methods For Employing the Anti-Inflammatory-Wound HealingCompositions of Embodiment Two (I.A-D+M4)

The present invention extends to methods for employing the therapeuticanti-inflammatory-wound healing compositions (I.A-D+M4). In general, atherapeutic composition is employed by contacting the therapeuticcomposition with a wound. In a preferred embodiment, the invention isdirected to a method for healing an inflamed wound in a mammal with ananti-inflammatory-wound healing composition (I.A+M4) which comprises thesteps of:

(A) providing a therapeutic anti-inflammatory-wound healing compositionwhich comprises:

(1) a therapeutically effective amount of an anti-inflammatory agent;and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) contacting the anti-inflammatory-wound healing composition with theinflamed wound.

d. Augmented Anti-inflammatory-Wound Healing Compositions of EmbodimentTwo (I.A-D+M4+M) and (I.A-D+M4+M)

In another aspect of Embodiment Two, the therapeuticanti-inflammatory-wound healing compositions (I.A-D+M4) of the presentinvention may be further combined with medicaments useful for treatingwounds (M) to form augmented anti-inflammatory-wound healingcompositions (I.A-D+M4+M). In this embodiment, the combination of theanti-inflammatory-wound healing composition of the present invention andthe medicament useful for treating wounds provides an augmentedanti-inflammatory-wound healing composition having an enhanced abilityto increase the proliferation and resuscitation rate of mammalian cells.For example, the therapeutic compositions of the present invention maybe used in combination with medicaments useful for treating wounds suchas immunostimulating agents (Betafectin™), antiviral agents,antikeratolytic agents, anti-other inflammatory agents, antifungalagents, tretinoin, sunscreen agents, dermatological agents, topicalantihistamine agents, antibacterial agents, bioadhesive agents,respiratory bursting inhibitors (lactic acid, adenosine), inhibitors ofprostaglandin synthesis (ibuprofen, aspirin, indomethacin, meclofenomicacid, retinoic acid, padimate O, meclomen, oxybenzone), steroidalanti-inflammatory agents (corticosteroids including synthetic analogs),antimicrobial agents (neosporin ointment, silvadine), antiseptic agents,anesthetic agents (pramoxine hydrochloride, lidocaine, benzocaine), cellnutrient media, burn relief medications, sun burn medications, acnepreparations, insect bite and sting medications, wound cleansers, wounddressings, scar reducing agents (vitamin E), and the like, and mixturesthereof, to further enhance the proliferation and resuscitation rate ofmammalian cells. Preferably, the medicament useful for treating woundsis selected from the group consisting of immunostimulating agents,antiviral agents, antikeratolytic agents, anti-inflammatory agents,antifungal agents, tretinoin, sunscreen agents, dermatological agents,topical antihistamine agents, antibacterial agents, bioadhesive agents,respiratory bursting inhibitors, inhibitors of prostaglandin synthesis,antimicrobial agents, cell nutrient media, scar reducing agents, andmixtures thereof. More preferably, the medicament useful for treatingwounds is selected from the group consisting of immunostimulatingagents, antiviral agents, antikeratolytic agents, anti-inflammatoryagents, antifungal agents, acne treating agents, sunscreen agents,dermatological agents, antihistamine agents, antibacterial agents,bioadhesive agents, and mixtures thereof.

In a preferred embodiment, the invention is directed to an augmentedanti-inflammatory-wound healing composition (I.A+M4+M) which comprises:

(A) a therapeutic anti-inflammatory-wound healing composition whichcomprises:

(1) a therapeutically effective amount of an anti-inflammatory agent;and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a medicament useful for treating wounds.

The present invention extends to methods for making the augmentedanti-inflammatory-wound healing compositions. In general, the augmentedcompositions are made by admixing the therapeuticanti-inflammatory-wound healing composition with the medicament usefulfor treating wounds to prepare the augmented anti-inflammatory-woundhealing composition.

The present invention also extends to methods for employing theaugmented anti-inflammatory-wound healing compositions. In general, anaugmented anti-inflammatory-wound healing composition is employed bycontacting the composition with a wound. In a preferred embodiment, theinvention is directed to a method for healing an inflamed wound in amammal with an augmented anti-inflammatory-wound healing composition(I.A+M4+M) which comprises the steps of:

(A) providing a therapeutic augmented anti-inflammatory-wound healingcomposition which comprises:

(1) a therapeutically effective amount of an anti-inflammatory agent;

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(3) providing a medicament useful for treating wounds; and

(B) contacting the augmented anti-inflammatory-wound healing compositionwith the inflamed wound.

The types of wounds which may be healed using theanti-inflammatory-wound healing compositions and the augmentedanti-inflammatory-wound healing compositions of the present inventionare those which result from an inflamed injury which causes epidermaldamage.

Methods for healing a wound comprise topically administering thecompositions of the present invention directly to a wound site toincrease the healing rate of the wound. The composition is maintained incontact with the wound for a period of time sufficient to increase theproliferation and resuscitation rate of the cells.

e. Formulations of the Anti-inflammatory-Wound Healing Compositions ofEmbodiment Two (I.A-D+M4) and (I.A-D+M4+M)

Once prepared, the inventive therapeutic anti-inflammatory-wound healingcompositions and augmented anti-inflammatory-wound healing compositionsmay be stored for future use or may be formulated in effective amountswith pharmaceutically acceptable carriers such as pharmaceuticalappliances and topical vehicles (oral and non-oral) to prepare a widevariety of pharmaceutical compositions. The pharmaceutically acceptablecarriers which may be employed and the methods used to prepare thepharmaceutical compositions have been described above in connection withthe formulations of the wound healing compositions of Embodiment One(I.A-D).

In a preferred embodiment, the invention is directed to ananti-inflammatory-wound healing pharmaceutical composition whichcomprises:

(A) a therapeutic anti-inflammatory-wound healing composition (I.A+M4)which comprises:

(1) a therapeutically effective amount of an anti-inflammatory agent;and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a pharmaceutically acceptable carrier selected from the groupconsisting of pharmaceutical appliances, bioadhesives, and occlusivevehicles.

In another preferred embodiment, the invention is directed to a methodfor preparing a pharmaceutical composition for increasing theproliferation and resuscitation rate of mammalian cells, which comprisesthe steps of:

(A) providing a therapeutically effective amount of ananti-inflammatory-wound healing composition (I.A+M4) which comprises:

(1) an anti-inflammatory agent; and

(2) a wound healing composition comprising:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(B) providing a pharmaceutically acceptable carrier; and

(C) admixing the anti-inflammatory-wound healing composition from step(A) and the pharmaceutically acceptable carrier from step (B) to form apharmaceutical composition.

5. Antifungal-Wound Healing Compositions a. Embodiment Two (I.A-D+M5)

Applicant has discovered therapeutic antifungal-wound healingcompositions (I.A-D+M5) which comprise a first antifungal agent (M5) andthe wound healing compositions of Embodiment One (I.A-D). Preferably,the wound healing composition (I.A) comprises (a) pyruvate, (b) anantioxidant, and (c) a mixture of saturated and unsaturated fatty acids.and a wound healing composition. The first antifungal agent is lacticacid or sorbic acid. Antifungal agents can treat fungal infections in apatient but do not promote the wound healing process. Wound healingcompositions can increase the resuscitation rate of injured mammaliancells and the proliferation rate of new mammalian cells to replace deadcells but do not treat fungal infections. Applicants have found that thecombination of an antifungal agent and a wound healing compositionresults in a therapeutic antifungal-wound healing composition whichreduces the duration and severity of fungal infections. The therapeuticantifungal-wound healing compositions may further comprise a secondantifungal agent and an anti-inflammatory agent.

In a preferred embodiment, the therapeutic antifungal-wound healingcompositions of the present invention may be administered to a patientin the form of a vaginal douche or a vaginal moisturizer, such as avaginal insert or suppository. Vaginal moisturizers, such as Replens™,relieve the discomfort of vaginal dryness. Vaginal moisturizers providerelief from the discomfort of vaginal dryness by providing continuoushydration to the vaginal tissue. Vaginal moisturizers typically containpurified water, glycerin mineral oil, polycarbophil, Carbomer 934P,hydrogenated palm oil glyceride, and sorbic acid. In this embodiment,the bioadhesive properties of the vaginal douche or moisturizer areemployed to deliver the antifungal agent and the wound healingcomposition to the vaginal walls to provide long lasting lubrication,antifungal treatment, and wound healing agents to facilitate healing forpostmenopausal women.

The combination of the first antifungal agent and the wound healingcompositions of the present invention provides a pharmaceuticalcomposition useful for treating fungal infections and having an enhancedability to prevent and reduce injury to mammalian cells and furtherincrease the resuscitation rate of injured mammalian cells. The tissuedamage associated with fungal infections is believed to be caused by theproduction of cellular produced active oxygen species. Combination ofthe first antifungal agent and the wound healing compositions maysuppress such reactive oxygen-linked tissue injury.

The first antifungal agent in the antifungal-wound healing compositionsof the present invention may be selected from the group consisting oflactic acid and sorbic acid. Preferably, the first antifungal agent islactic acid. More preferably, the first antifungal agent is a mixture oflactic acid and sorbic acid. Lactic acid (2-hydroxypropanoic acid)promotes the growth of lactobacilli and inhibits the growth ofpathogens. Sorbic acid (2,4-hexadienoic acid) is a natural antifungalagent which kills Candida albicans. Sorbic acid and lactic acid aresoluble in water.

The first antifungal agent of the present invention may be used in manydistinct physical forms well known in the pharmaceutical art to providean initial dosage of the first antifungal agent and/or a furthertime-release form of the first antifungal agent. Without being limitedthereto, such physical forms include free forms and encapsulated forms,and mixtures thereof.

The amount of first antifungal agent used in the present invention mayvary depending upon the therapeutic dosage recommended or permitted forthe particular first antifungal agent. In general, the amount of firstantifungal agent present is the ordinary dosage required to obtain thedesired result. Such dosages are known to the skilled practitioner inthe medical arts and are not a part of the present invention. In apreferred embodiment, the first antifungal agent in the antifungal-woundhealing composition is present in an amount from about 0.05% to about10%, preferably from about 0.1% to about 5%, and more preferably fromabout 0.2% to about 4%, by weight.

The therapeutic antifungal-wound healing compositions of the presentinvention may further comprise a second antifungal agent. The secondantifungal agent may be selected from a wide variety of water-solubleand water-insoluble drugs and their acid addition or metallic salts.Both organic and inorganic salts may be used provided the secondantifungal agent maintains its medicament value. The second antifungalagent may be selected from a wide range of therapeutic agents andmixtures of therapeutic agents which may be administered in sustainedrelease or prolonged action form. Nonlimiting illustrative specificexamples of antifungal agents include the following medicaments:miconazole, clotrimazole, tioconazole, terconazole, povidone-iodine, andbutoconazole. Preferred second antifungal agents to be employed may beselected from the group consisting of miconazole and clotrimazole.

The second antifungal agent of the present invention may be used in manydistinct physical forms well known in the pharmaceutical art to providean initial dosage of the second antifungal agent and/or a furthertime-release form of the second antifungal agent. Without being limitedthereto, such physical forms include free forms and encapsulated forms,and mixtures thereof.

The amount of second antifungal agent used in the present invention mayvary depending upon the therapeutic dosage recommended or permitted forthe particular second antifungal agent. In general, the amount of secondantifungal agent present is the ordinary dosage required to obtain thedesired result. Such dosages are known to the skilled practitioner inthe medical arts and are not a part of the present invention. In apreferred embodiment, the second antifungal agent in theantifungal-wound healing composition is present in an amount from about0.05% to about 10%, preferably from about 0.1% to about 5%, and morepreferably from about 0.2% to about 4%, by weight.

The therapeutic antifungal-wound healing compositions of the presentinvention may further comprise an anti-inflammatory agent. Theanti-inflammatory agent may be selected from a wide variety ofwater-soluble and water-insoluble drugs and their acid addition ormetallic salts. Both organic and inorganic salts may be used providedthe anti-inflammatory agent maintains its medicament value. Theanti-inflammatory agent may be selected from a wide range of therapeuticagents and mixtures of therapeutic agents which may be administered insustained release or prolonged action form. Nonlimiting illustrativespecific examples of non-steroidal anti-inflammatory agents include thefollowing medicaments: ibuprofen, naproxen, sulindac, diflunisal,piroxicam; indomethacin, etodolac, meclofenamate sodium; fenoprobencalcium, ketoprofen, mefenamic acid, nabumetone, ketorolac tromethamine,diclofenac, and evening primrose oil (containing about 72% linoleic acidand about 9% gamma-linolenic acid). Nonlimiting illustrative specificexamples of salicylate anti-inflammatory agents include the followingmedicaments: acetylsalicylic acid, mesalamine, salsalate, diflunisal,salicylsalicylic acid, and choline magnesia trisalicylate. Nonlimitingillustrative specific examples of steroidal anti-inflammatory agentsinclude the following medicaments: flunisolide, triamcinoline,triamcinoline acetonide, beclomethasone diproprionate, betamethasonediproprionate, hydrocortisone, cortisone, dexamethasone, prednisone,methyl prednisolone, and prednisolone.

The amount of anti-inflammatory agent used in the present invention mayvary depending upon the therapeutic dosage recommended or permitted forthe particular anti-inflammatory agent. In general, the amount ofanti-inflammatory agent present is the ordinary dosage required toobtain the desired result. Such dosages are known to the skilledpractitioner in the medical arts and are not a part of the presentinvention. In a preferred embodiment, the anti-inflammatory agent in theantifungal-wound healing composition is present in an amount from about0.05% to about 10%, preferably from about 0.1% to about 5%, and morepreferably from about 0.2% to about 4%, by weight.

b. Methods For Making the Antifungal-Wound Healing Compositions ofEmbodiment Two (I.A-D+M5)

The present invention extends to methods for making the therapeuticantifungal-wound healing compositions (I.A-D+M5). In general, atherapeutic antifungal-wound healing composition is made by forming anadmixture of the wound healing components of Embodiment One (I.A-D) andan antifungal agent. In a first aspect of Embodiment Two (I.A+M5), anantifungal-wound healing therapeutic composition is made by forming anadmixture of an antifungal agent and a wound healing compositioncomprising (a) a pyruvate, (b) an antioxidant, and (c) a mixture ofsaturated and unsaturated fatty acids. In a second aspect of EmbodimentTwo (I.B+M5), an antifungal-wound healing therapeutic composition ismade by forming an admixture of an antifungal agent and a wound healingcomposition comprising (a) a pyruvate, (b) a lactate, and (c) a mixtureof saturated and unsaturated fatty acids. In a third aspect ofEmbodiment Two (I.C+M5), an antifungal-wound healing therapeuticcomposition is made by forming an admixture of an antifungal agent and awound healing composition comprising (a) an antioxidant, and (b) amixture of saturated and unsaturated fatty acids. In a fourth aspect ofEmbodiment Two (I.D+M5), an antifungal-wound healing therapeuticcomposition is made by forming an admixture of an antifungal agent and awound healing composition comprising (a) a lactate, (b) an antioxidant,and (c) a mixture of saturated and unsaturated fatty acids.

In a preferred embodiment, the invention is directed to a method forpreparing a therapeutic antifungal-wound healing composition (I.A+M5)which comprises the steps of admixing the following ingredients:

(A) a therapeutically effective amount of an antifungal agent; and

(B) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

c. Methods For Employing the Antifungal-Wound Healing Compositions ofEmbodiment Two (I.A-D+M5)

The present invention extends to methods for employing the therapeuticantifungal-wound healing compositions (I.A-D+M5). In general, atherapeutic composition is employed by contacting the therapeuticcomposition with a wound. In a preferred embodiment, the invention isdirected to a method for healing a infected wound in a mammal with anantifungal-wound healing composition (I.A+M5) which comprises the stepsof:

(A) providing a therapeutic antifungal-wound healing composition whichcomprises:

(1) a therapeutically effective amount of an antifungal agent; and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) contacting the antifungal-wound healing composition with theinfected wound.

d. Augmented Antifungal-Wound Healing Compositions of Embodiment Two(I.A-D+M5+M)

In another aspect of Embodiment Two, the therapeutic antifungal-woundhealing compositions (I.A-D+M5) of the present invention may be furthercombined with medicaments useful for treating wounds (M) to formaugmented antifungal-wound healing compositions (I.A-D+M5+M). In thisembodiment, the combination of the antifungal-wound healing compositionof the present invention and the medicament useful for treating woundsprovides an augmented antifungal-wound healing composition having anenhanced ability to increase the proliferation and resuscitation rate ofmammalian cells. For example, the therapeutic compositions of thepresent invention may be used in combination with medicaments useful fortreating wounds such as immunostimulating agents (Betafectin™),antiviral agents, antikeratolytic agents, anti-inflammatory agents,other antifungal agents, tretinoin, sunscreen agents, dermatologicalagents, topical antihistamine agents, antibacterial agents, bioadhesiveagents, respiratory bursting inhibitors (lactic acid, adenosine),inhibitors of prostaglandin synthesis (ibuprofen, aspirin, indomethacin,meclofenomic acid, retinoic acid, padimate O, meclomen, oxybenzone),steroidal anti-inflammatory agents (corticosteroids including syntheticanalogs), antimicrobial agents (neosporin ointment, silvadine),antiseptic agents, anesthetic agents (pramoxine hydrochloride,lidocaine, benzocaine), cell nutrient media, burn relief medications,sun burn medications, acne preparations, insect bite and stingmedications, wound cleansers, wound dressings, scar reducing agents(vitamin E), and the like, and mixtures thereof, to further enhance theproliferation and resuscitation rate of mammalian cells. Preferably, themedicament useful for treating wounds is selected from the groupconsisting of immunostimulating agents, antiviral agents,antikeratolytic agents, anti-inflammatory agents, antifungal agents,tretinoin, sunscreen agents, dermatological agents, topicalantihistamine agents, antibacterial agents, bioadhesive agents,respiratory bursting inhibitors, inhibitors of prostaglandin synthesis,antimicrobial agents, cell nutrient media, scar reducing agents, andmixtures thereof. More preferably, the medicament useful for treatingwounds is selected from the group consisting of immunostimulatingagents, antiviral agents, antikeratolytic agents, anti-inflammatoryagents, antifungal agents, acne treating agents, sunscreen agents,dermatological agents, antihistamine agents, antibacterial agents,bioadhesive agents, and mixtures thereof.

In a preferred embodiment, the invention is directed to an augmentedantifungal-wound healing composition (I.A+M5+M) which comprises:

(A) a therapeutic antifungal-wound healing composition which comprises:

(1) a therapeutically effective amount of an antifungal agent; and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a medicament useful for treating wounds.

The present invention extends to methods for making the augmentedantifungal-wound healing compositions. In general, the augmentedcompositions are made by admixing the therapeutic antifungal-woundhealing composition with the medicament useful for treating wounds toprepare the augmented antifungal-wound healing composition.

The present invention also extends to methods for employing theaugmented antifungal-wound healing compositions. In general, anaugmented antifungal-wound healing composition is employed by contactingthe composition with a wound. In a preferred embodiment, the inventionis directed to a method for healing an infected wound in a mammal withan augmented antifungal-wound healing composition (I.A+M5+M) whichcomprises the steps of:

(A) providing a therapeutic augmented antifungal-wound healingcomposition which comprises:

(1) a therapeutically effective amount of an antifungal agent;

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(3) providing a medicament useful for treating wounds; and

(B) contacting the augmented antifungal-wound healing composition withthe infected wound.

The types of wounds which may be healed using the antifungal-woundhealing compositions and the augmented antifungal-wound healingcompositions of the present invention are those which result from aninfected injury which causes epidermal damage.

Methods for healing a wound comprise topically administering thecompositions of the present invention directly to a wound site toincrease the healing rate of the wound. The composition is maintained incontact with the wound for a period of time sufficient to increase theproliferation and resuscitation rate of the cells.

e. Formulations of the Antifungal-Wound Healing Compositions ofEmbodiment Two (I.A-D+M5) and (I.A-D+M5+M)

Once prepared, the inventive therapeutic antifungal-wound healingcompositions and augmented antifungal-wound healing compositions may bestored for future use or may be formulated in effective amounts withpharmaceutically acceptable carriers such as pharmaceutical appliancesand topical vehicles (oral and non-oral) to prepare a wide variety ofpharmaceutical compositions. The pharmaceutically acceptable carrierswhich may be employed and the methods used to prepare the pharmaceuticalcompositions have been described above in connection with theformulations of the wound healing compositions of Embodiment One(I.A-D).

In a preferred embodiment, the invention is directed to anantifungal-wound healing pharmaceutical composition which comprises:

(A) a therapeutic antifungal-wound healing composition (I.A+M5) whichcomprises:

(1) a therapeutically effective amount of an antifungal agent; and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a pharmaceutically acceptable carrier selected from the groupconsisting of pharmaceutical appliances, bioadhesives, and occlusivevehicles.

In another preferred embodiment, the invention is directed to a methodfor preparing a pharmaceutical composition for increasing theproliferation and resuscitation rate of mammalian cells, which comprisesthe steps of:

(A) providing a therapeutically effective amount of an antifungal-woundhealing composition (I.A+M5) which comprises:

(1) an antifungal agent; and

(2) a wound healing composition comprising:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(B) providing a pharmaceutically acceptable carrier; and

(C) admixing the antifungal-wound healing composition from step (A) andthe pharmaceutically acceptable carrier from step (B) to form apharmaceutical composition.

6. Acne Treating-Wound Healing Compositions a. Embodiment Two (I.A-D+M6)

Applicant has discovered therapeutic acne treating-wound healingcompositions (I.A-D+M6) which comprise tretinoin (M6) and the woundhealing compositions of Embodiment One (I.A-D). Preferably, the woundhealing composition (I.A) comprises (a) pyruvate, (b) an antioxidant,and (c) a mixture of saturated and unsaturated fatty acids. Tretinoin isuseful for the topical treatment of acne vulgaris but is known to induceexcessive redness, edematous blistering or crusting, and severe localerythema and peeling at the site of application. Wound healingcompositions can increase the resuscitation rate of injured mammaliancells and the proliferation rate of new mammalian cells to replace deadcells but do not treat acne vulgaris. Applicants have found that thecombination of tretinoin and a wound healing composition results in atherapeutic acne treating-wound healing composition which reduces theduration and severity of acne vulgaris, as an augmented wound healingcomposition, and the irritation associated with tretinoin, as acytoprotective-wound healing composition. This invention also relates tomethods for employing the therapeutic acne treating-wound healingcompositions to treat wrinkles.

The combination of tretinoin and the wound healing compositions of thepresent invention provides a pharmaceutical composition useful fortreating acne vulgaris and having an enhanced ability to prevent andreduce injury to mammalian cells and further increase the resuscitationrate of injured mammalian cells. The tissue damage associated withtretinoin induced inflammation is believed to be caused by theproduction of cellular produced active oxygen species. Combination oftretinoin and the wound healing compositions may suppress such reactiveoxygen-linked tissue injury.

As set out above, tretinoin (Retin-A™) is indicated for the topicaltreatment of acne vulgaris. Chemically, tretinoin is all-trans-retinoicacid (vitamin A acid). Tretinoin is available in a gel vehicleconsisting of butylated hydroxytoluene, hydroxypropyl cellulose, andalcohol; in a hydrophilic cream vehicle consisting of stearic acid,isopropyl myristate, polyoxyl 40 stearate, stearyl alcohol, xanthan gum,sorbic acid, butylated hydroxytoluene, and purified water; and in aliquid vehicle consisting of polyethylene glycol 400, butylatedhydroxytoluene, and alcohol. Tretinoin may be used in many distinctphysical forms well known in the pharmaceutical art to provide aninitial dosage of tretinoin and/or a further time-release form oftretinoin. Without being limited thereto, such physical forms includefree forms and encapsulated forms, and mixtures thereof.

The amount of tretinoin used in the present invention may vary dependingupon the therapeutic dosage recommended or permitted for the particularpatient. In general, the amount of tretinoin present is the ordinarydosage required to obtain the desired result. Such dosages are known tothe skilled practitioner in the medical arts and are not a part of thepresent invention. In a preferred embodiment, tretinoin in the acnetreating-wound healing composition is present in an amount from about0.01% to about 0.1%, and preferably from about 0.025% to about 0.05%, byweight.

b. Methods For Making the Acne Treating-Wound Healing Compositions ofEmbodiment Two (I.A-D+M6)

The present invention extends to methods for making the therapeutic acnetreating-wound healing compositions (I.A-D+M6). In general, atherapeutic acne treating-wound healing composition is made by formingan admixture of the wound healing components of Embodiment One (I.A-D)and tretinoin. In a first aspect of Embodiment Two (I.A+M6), an acnetreating-wound healing therapeutic composition is made by forming anadmixture of tretinoin and a wound healing composition comprising (a) apyruvate, (b) an antioxidant, and (c) a mixture of saturated andunsaturated fatty acids. In a second aspect of Embodiment Two (I.B+M6),an acne treating-wound healing therapeutic composition is made byforming an admixture of tretinoin and a wound healing compositioncomprising (a) a pyruvate, (b) a lactate, and (c) a mixture of saturatedand unsaturated fatty acids. In a third aspect of Embodiment Two(I.C+M6), an acne treating-wound healing therapeutic composition is madeby forming an admixture of tretinoin and a wound healing compositioncomprising (a) an antioxidant, and (b) a mixture of saturated andunsaturated fatty acids. In a fourth aspect of Embodiment Two (I.D+M6),an acne treating-wound healing therapeutic composition is made byforming an admixture of tretinoin and a wound healing compositioncomprising (a) a lactate, (b) an antioxidant, and (c) a mixture ofsaturated and unsaturated fatty acids.

In a preferred embodiment, the invention is directed to a method forpreparing a therapeutic acne treating-wound healing composition (I.A+M6)which comprises the steps of admixing the following ingredients:

(A) a therapeutically effective amount of tretinoin; and

(B) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

c. Methods For Employing the Acne Treating-Wound Healing Compositions ofEmbodiment Two (I.A-D+M6)

The present invention extends to methods for employing the therapeuticacne treating-wound healing compositions (I.A-D+M6). In general, atherapeutic composition is employed by contacting the therapeuticcomposition with acne vulgaris. In a preferred embodiment, the inventionis directed to a method for treating acne vulgaris in a human with anacne treating-wound healing composition (I.A+M6) which comprises thesteps of:

(A) providing a therapeutic acne treating-wound healing compositionwhich comprises:

(1) a therapeutically effective amount of tretinoin; and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) contacting the acne treating-wound healing composition with theinfected wound.

In another preferred embodiment, the present invention extends tomethods for employing the therapeutic acne treating-wound healingcompositions (I.A-D+M6) to treat wrinkles. Tretinoin is also used todecrease wrinkles because it decreases the cohesiveness of follicularepithelial cells with decreased microcomedo formation. In general, atherapeutic composition is employed by contacting the therapeuticcomposition with the wrinkles. In a specific embodiment, the inventionis directed to a method for treating wrinkles in a human with an acnetreating-wound healing composition (I.A-D+M6) which comprises the stepsof:

(A) providing a therapeutic acne treating-wound healing compositionwhich comprises:

(1) a therapeutically effective amount of tretinoin; and

(2) a wound healing composition comprising:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) contacting the acne treating-wound healing composition with thewrinkles.

The types of wounds which may be healed using the acne treating-woundhealing compositions and the augmented acne treating-wound healingcompositions of the present invention are acne vulgaris.

Methods for treating acne vulgaris or wrinkles comprise topicallyadministering the compositions of the present invention directly to theacne vulgaris or wrinkles. The composition is maintained in contact withthe acne vulgaris or wrinkles for a period of time sufficient toincrease the proliferation and resuscitation rate of the cells.

d. Augmented Acne Treating-Wound Healing Compositions of Embodiment Two(I.A-D+M6+M)

In another aspect of Embodiment Two, the therapeutic acne treating-woundhealing compositions (I.A-D+M6) of the present invention may be furthercombined with medicaments useful for treating wounds (M) to formaugmented acne treating-wound healing compositions (I.A-D+M6+M). In thisembodiment, the combination of the acne treating-wound healingcomposition of the present invention and the medicament useful fortreating wounds provides an augmented acne treating-wound healingcomposition having an enhanced ability to increase the proliferation andresuscitation rate of mammalian cells. For example, the therapeuticcompositions of the present invention may be used in combination withmedicaments useful for treating wounds such as immunostimulating agents(Betafectin™), antiviral agents, antikeratolytic agents,anti-inflammatory agents, antifungal agents, tretinoin, sunscreenagents, dermatological agents, topical antihistamine agents,antibacterial agents, bioadhesive agents, respiratory burstinginhibitors (lactic acid, adenosine), inhibitors of prostaglandinsynthesis (ibuprofen, aspirin, indomethacin, meclofenomic acid, retinoicacid, padimate O, meclomen, oxybenzone), steroidal anti-inflammatoryagents (corticosteroids including synthetic analogs), antimicrobialagents (neosporin ointment, silvadine), antiseptic agents, anestheticagents (pramoxine hydrochloride, lidocaine, benzocaine), cell nutrientmedia, burn relief medications, sun burn medications, acne preparations,insect bite and sting medications, wound cleansers, wound dressings,scar reducing agents (vitamin E), and the like, and mixtures thereof, tofurther enhance the proliferation and resuscitation rate of mammaliancells. Preferably, the medicament useful for treating wounds is selectedfrom the group consisting of immunostimulating agents, antiviral agents,antikeratolytic agents, anti-inflammatory agents, antifungal agents,tretinoin, sunscreen agents, dermatological agents, topicalantihistamine agents, antibacterial agents, bioadhesive agents,respiratory bursting inhibitors, inhibitors of prostaglandin synthesis,antimicrobial agents, cell nutrient media, scar reducing agents, andmixtures thereof. More preferably, the medicament useful for treatingwounds is selected from the group consisting of immunostimulatingagents, antiviral agents, antikeratolytic agents, anti-inflammatoryagents, antifungal agents, acne treating agents, sunscreen agents,dermatological agents, antihistamine agents, antibacterial agents,bioadhesive agents, and mixtures thereof.

In a preferred embodiment, the invention is directed to an augmentedacne treating-wound healing composition (I.A+M6+M) which comprises:

(A) a therapeutic acne treating-wound healing composition whichcomprises: (1) a therapeutically effective amount of tretinoin; and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a medicament useful for treating wounds.

The present invention extends to methods for making the augmented acnetreating-wound healing compositions. In general, the augmentedcompositions are made by admixing the therapeutic acne treating-woundhealing composition with the medicament useful for treating wounds toprepare the augmented acne treating-wound healing composition.

The present invention extends to methods for employing the therapeuticaugmented acne treating-wound healing compositions. In general, atherapeutic augmented composition is employed by contacting thetherapeutic composition with the acne vulgaris. In a preferredembodiment, the invention is directed to a method for treating acnevulgaris in a human with an augmented acne treating-wound healingcomposition acne treating-wound healing composition (I.A+M6+M) whichcomprises the steps of:

(A) providing a therapeutic augmented acne treating-wound healingcomposition which comprises:

(1) a therapeutically effective amount of tretinoin;

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(3) providing a medicament useful for treating wounds; and

(B) contacting the acne treating-wound healing composition with the acnevulgaris.

In another preferred embodiment, the present invention extends tomethods for employing the therapeutic augmented acne treating-woundhealing compositions to treat wrinkles. In general, a therapeuticaugmented composition is employed by contacting the therapeuticcomposition with the wrinkles. In a specific embodiment, the inventionis directed to a method for treating wrinkles in a human with anaugmented acne treating-wound healing composition (I.A+M6+M) whichcomprises the steps of:

(A) providing a therapeutic acne treating-wound healing compositionwhich comprises:

(1) a therapeutically effective amount of tretinoin; and

(2) a wound healing composition comprising:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) contacting the acne treating-wound healing composition with thewrinkles.

e. Formulations of the Acne Treating-Wound Healing Compositions ofEmbodiment Two (I.A-D+M6) and (I.A-D+M6+M)

Once prepared, the inventive therapeutic acne treating-wound healingcompositions and augmented acne treating-wound healing compositions maybe stored for future use or may be formulated in effective amounts withpharmaceutically acceptable carriers such as pharmaceutical appliancesand topical vehicles (oral and non-oral) to prepare a wide variety ofpharmaceutical compositions. The pharmaceutically acceptable carrierswhich may be employed and the methods used to prepare the pharmaceuticalcompositions have been described above in connection with theformulations of the wound healing compositions of Embodiment One(I.A-D).

In a preferred embodiment, the invention is directed to an acnetreating-wound healing pharmaceutical composition which comprises:

(A) a therapeutic acne treating-wound healing composition (I.A+M6) whichcomprises:

(1) a therapeutically effective amount of tretinoin; and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a pharmaceutically acceptable carrier selected from the groupconsisting of pharmaceutical appliances, bioadhesives, and occlusivevehicles.

In another preferred embodiment, the invention is directed to a methodfor preparing a pharmaceutical composition for increasing theproliferation and resuscitation rate of mammalian cells, which comprisesthe steps of:

(A) providing a therapeutically effective amount of an acnetreating-wound healing composition (I.A+M6) which comprises:

(1) tretinoin; and

(2) a wound healing composition comprising:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(B) providing a pharmaceutically acceptable carrier; and

(C) admixing the acne treating-wound healing composition from step (A)and the pharmaceutically acceptable carrier from step (B) to form apharmaceutical composition.

7. Sunscreen Wound Healing Compositions a. Embodiment Two (I.A-D+M7)

Applicant has discovered therapeutic sunscreen-wound healingcompositions (I.A-D+M7) which comprise a therapeutically effectiveamount of a sunscreen agent and an anti-inflammatory agent (collectivelyreferred to as M7) and the wound healing compositions of Embodiment One(I.A-D). Preferably, the wound healing composition (I.A) comprises (a)pyruvate, (b) an antioxidant, and (c) a mixture of saturated andunsaturated fatty acids. Sunscreen agents can help prevent sunburn byscreening ultra violet light but do not heal injured mammalian cells.Anti-inflammatory agents can reduce inflammation (erythema) in a patientbut do not promote the wound healing process. Wound healing compositionscan increase the resuscitation rate of injured mammalian cells and theproliferation rate of new mammalian cells to replace dead cells. Woundhealing compositions can also minimize oxygen radical damage from ultraviolet light. Applicants have found that the combination of a sunscreenagent, an anti-inflammatory agent, and a wound healing compositionresults in a therapeutic sunscreen-wound healing compositions useful forminimizing and treating sunburn damage. The sunscreen-wound healingcompositions may optionally contain a therapeutically effective amountof a topical anesthetic to further reduce the duration and severity ofsunburn.

The combination of the sunscreen agent, the anti-inflammatory agent, andthe wound healing compositions of the present invention provides apharmaceutical composition useful for minimizing and treating sunburndamage and having an enhanced ability to prevent and reduce injury tomammalian cells and further increase the resuscitation rate of injuredmammalian cells. The tissue damage associated with sunburn is believedto be caused by the production of cellular produced active oxygenspecies. Combination of the sunscreen agent, anti-inflammatory agent,and the wound healing compositions helps suppress such reactiveoxygen-linked tissue injury.

Sunscreen agents are compounds which provide broad spectrum protectionfrom ultra violet A and ultra violet B light from the sun. The sunscreenagents in the sunscreen-wound healing compositions of the presentinvention may be selected from a wide range of therapeutic agents andmixtures of therapeutic agents. Nonlimiting illustrative specificexamples of sunscreen agents include ethylhexyl p-methoxycinnamate,octyl methoxycinnamate, octyl dimethyl p-aminobenzoic acid, 2-ethylhexylsalicylate, octyl salicylate, menthyl anthranilate, octocrylene,padimate o, titanium dioxide, urea, and oxybenzone. Preferably, thesunscreen agent is oxybenzone.

The amount of sunscreen agent used in the present invention is atherapeutically effective amount and may vary depending upon thetherapeutic dosage recommended or permitted for the particular sunscreenagent. In general, the amount of sunscreen agent present is the ordinarydosage required to obtain the desired result. Such dosages are known tothe skilled practitioner in the medical arts and are not a part of thepresent invention. In a preferred embodiment, the sunscreen agent in thesunscreen-wound healing composition is present in an amount from about1% to about 30%, preferably from about 2% to about 25%, and morepreferably from about 4% to about 20%, by weight.

Anti-inflammatory agents are compounds that counteract or suppress theinflammatory process. The anti-inflammatory agents in thesunscreen-wound healing compositions of the present invention may beselected from a wide variety of steroidal, non-steroidal, and salicylatewater-soluble and water-insoluble drugs and their acid addition ormetallic salts. Both organic and inorganic salts may be used providedthe anti-inflammatory agent maintains its medicament value. Theanti-inflammatory agents may be selected from a wide range oftherapeutic agents and mixtures of therapeutic agents which may beadministered in sustained release or prolonged action form. Nonlimitingillustrative specific examples of non-steroidal anti-inflammatory agentsinclude the following medicaments: ibuprofen, naproxen, sulindac,diflunisal, piroxicam, indomethacin, etodolac, meclofenamate sodium,fenoproben calcium, ketoprofen, mefenamic acid, nabumetone, ketorolactromethamine, diclofenac, and evening primrose oil (containing about 72%linoleic acid and about 9% gamma-linolenic acid). Nonlimitingillustrative specific examples of salicylate anti-inflammatory agentsinclude the following medicaments: acetylsalicylic acid, mesalamine,salsalate, diflunisal, salicylsalicylic acid, and choline magnesiumtrisalicylate. Nonlimiting illustrative specific examples of steroidalanti-inflammatory agents include the following medicaments: flunisolide,triamcinoline, triamcinoline acetonide, beclomethasone diproprionate,betamethasone diproprionate, hydrocortisone, cortisone, dexamethasone,prednisone, methyl prednisolone, and prednisolone.

Preferred anti-inflammatory agents to be employed may be selected fromthe group consisting of ibuprofen, naproxen, sulindac, diflunisal,piroxicam, indomethacin, etodolac, meclofenamate sodium, fenoprobencalcium, ketoprofen, mefenamic acid, nabumetone, ketorolac tromethamine,diclofenac, evening primrose oil, acetylsalicylic acid, mesalamine,salsalate, diflunisal, salicylsalicylic acid, choline magnesiumtrisalicylate, flunisolide, triamcinoline, triamcinoline acetonide,beclomethasone diproprionate, betamethasone diproprionate,hydrocortisone, cortisone, dexamethasone, prednisone, methylprednisolone, and prednisolone. In a preferred embodiment, theanti-inflammatory agent is selected from the group consisting ofibuprofen, naproxen, sulindac, diflunisal, piroxicam, indomethacin,etodolac, meclofenamate sodium, fenoproben calcium, ketoprofen,mefenamic acid, nabumetone, ketorolac tromethamine, diclofenac, andevening primrose oil. In a more preferred embodiment, theanti-inflammatory agent is evening primrose oil.

The anti-inflammatory agent of the present invention may be used in manydistinct physical forms well known in the pharmaceutical art to providean initial dosage of the anti-inflammatory agent and/or a furthertime-release form of the anti-inflammatory agent. Without being limitedthereto, such physical forms include free forms and encapsulated forms,and mixtures thereof.

The amount of anti-inflammatory agent used in the present invention is atherapeutically effective amount and may vary depending upon thetherapeutic dosage recommended or permitted for the particularanti-inflammatory agent. In general, the amount of anti-inflammatoryagent present is the ordinary dosage required to obtain the desiredresult. Such dosages are known to the skilled practitioner in themedical arts and are not a part of the present invention. In a preferredembodiment, the anti-inflammatory agent in the sunscreen-wound healingcomposition is present in an amount from about 0.01% to about 10%,preferably from about 0.1% to about 5%, and more preferably from about1% to about 3%, by weight.

In yet another preferred embodiment, the therapeutic sunscreen-woundhealing compositions of the present invention further comprise a topicalanesthetic agent. Anesthetic agents are compounds that induce the lossof tactile sensibility and the sensation of pain. The anesthetic agentsin the sunscreen-wound healing compositions of the present invention maybe selected from a wide range of therapeutic agents and mixtures oftherapeutic agents. Nonlimiting illustrative specific examples oftopical anesthetic agents include pramoxine hydrochloride, lidocaine,and benzocaine.

The amount of anesthetic agent used in the present invention is atherapeutically effective amount and may vary depending upon thetherapeutic dosage recommended or permitted for the particularanesthetic agents. In general, the amount of anesthetic agents presentis the ordinary dosage required to obtain the desired result. Suchdosages are known to the skilled practitioner in the medical arts andare not a part of the present invention. In a preferred embodiment, theanesthetic agent in the sunscreen-wound healing composition is presentin an amount from about 1% to about 30%, preferably from about 2% toabout 25%, and more preferably from about 2.5% to about 20%, by weight.

b. Methods For Making the Sunscreen-Wound Healing Compositions ofEmbodiment Two (I.A-D+M7)

The present invention extends to methods for making the therapeuticsunscreen-wound healing compositions (I.A-D+M7). In general, atherapeutic sunscreen-wound healing composition is made by forming anadmixture of the wound healing components of Embodiment One (I.A-D), asunscreen agent, and an anti-inflammatory agent. In a first aspect ofEmbodiment Two (I.A+M7), a sunscreen-wound healing therapeuticcomposition is made by forming an admixture of a sunscreen agent, ananti-inflammatory agent, and a wound healing composition comprising (a)a pyruvate, (b) an antioxidant, and (c) a mixture of saturated andunsaturated fatty acids. In a second aspect of Embodiment Two (I.B+M7),a sunscreen-wound healing therapeutic composition is made by forming anadmixture of a sunscreen agent, an anti-inflammatory agent, and a woundhealing composition comprising (a) a pyruvate, (b) a lactate, and (c) amixture of saturated and unsaturated fatty acids. In a third aspect ofEmbodiment Two (I.C+M7), a sunscreen-wound healing therapeuticcomposition is made by forming an admixture of a sunscreen agent, ananti-inflammatory agent, and a wound healing composition comprising (a)an antioxidant, and (b) a mixture of saturated and unsaturated fattyacids. In a fourth aspect of Embodiment Two (I.D+M7), a sunscreen-woundhealing therapeutic composition is made by forming an admixture ofadmixture of a sunscreen agent, an anti-inflammatory agent, and a woundhealing composition comprising (a) a lactate, (b) an antioxidant, and(c) a mixture of saturated and unsaturated fatty acids.

In a preferred embodiment, the invention is directed to a method forpreparing a therapeutic sunscreen-wound healing composition (I.A+M7)useful to minimize and treat sunburn damage which comprises the steps ofadmixing a therapeutically effective amount of the followingingredients:

(1) a sunscreen agent;

(2) an anti-inflammatory agent; and

(3) a wound healing composition comprising:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

c. Methods for Employing the Sunscreen-Wound Healing Compositions ofEmbodiment Two (I.A-D+M7)

The present invention extends to methods for employing the therapeuticsunscreen-wound healing compositions (I.A-D+M7). In general, atherapeutic composition is employed by contacting the therapeuticcomposition with the skin to be exposed to the sun. In a preferredembodiment, the invention is directed to a method for minimizing andtreating sunburn in a human with a sunscreen-wound healing composition(I.A+M7) which comprises the steps of:

(A) providing a therapeutically effective amount of a sunscreen-woundhealing composition which comprises:

(1) a sunscreen agent;

(2) an anti-inflammatory agent; and

(3) a wound healing composition comprising:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) contacting the sunscreen-wound healing composition with the skin ofthe human prior to exposure to the sun.

d. Augmented Sunscreen-Wound Healing Compositions of Embodiment Two(I.A-D+M7+M)

In another aspect of Embodiment Two, the therapeutic sunscreen-woundhealing compositions (I.A-D+M7) of the present invention may be furthercombined with medicaments useful for treating wounds (M) to formaugmented sunscreen-wound healing compositions (I.A-D+M7+M). In thisembodiment, the combination of the sunscreen-wound healing compositionof the present invention and the medicament useful for treating woundsprovides an augmented sunscreen-wound healing composition having anenhanced ability to increase the proliferation and resuscitation rate ofmammalian cells. For example, the therapeutic compositions of thepresent invention may be used in combination with medicaments useful fortreating wounds such as immunostimulating agents (Betafectin™),antiviral agents, antikeratolytic agents, anti-inflammatory agents,antifungal agents, tretinoin, other sunscreen agents, dermatologicalagents, topical antihistamine agents, antibacterial agents, bioadhesiveagents, respiratory bursting inhibitors (lactic acid, adenosine),inhibitors of prostaglandin synthesis (ibuprofen, aspirin, indomethacin,meclofenomic acid, retinoic acid, padimate O, meclomen, oxybenzone),steroidal anti-inflammatory agents (corticosteroids including syntheticanalogs), antimicrobial agents (neosporin ointment, silvadine),antiseptic agents, anesthetic agents (pramoxine hydrochloride,lidocaine, benzocaine), cell nutrient media, burn relief medications,sun burn medications, acne preparations, insect bite and stingmedications, wound cleansers, wound dressings, scar reducing agents(vitamin E), and the like, and mixtures thereof, to further enhance theproliferation and resuscitation rate of mammalian cells. Preferably, themedicament useful for treating wounds is selected from the groupconsisting of immunostimulating agents, antiviral agents,antikeratolytic agents, anti-inflammatory agents, antifungal agents,tretinoin, sunscreen agents, dermatological agents, topicalantihistamine agents, antibacterial agents, bioadhesive agents,respiratory bursting inhibitors, inhibitors of prostaglandin synthesis,antimicrobial agents, cell nutrient media, scar reducing agents, andmixtures thereof. More preferably, the medicament useful for treatingwounds is selected from the group consisting of immunostimulatingagents, antiviral agents, antikeratolytic agents, anti-inflammatoryagents, antifungal agents, acne treating agents, sunscreen agents,dermatological agents, antihistamine agents, antibacterial agents,bioadhesive agents, and mixtures thereof.

In a preferred embodiment, the invention is directed to an augmentedsunscreen-wound healing composition (I.A+M7+M) useful to minimize andtreat sunburn damage which comprises:

(A) a therapeutic sunscreen-wound healing composition which comprises atherapeutically effective amount of:

(1) a sunscreen agent;

(2) an anti-inflammatory agent; and

(3) a wound healing composition, wherein the wound healing compositioncomprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a medicament useful for treating wounds.

The present invention extends to methods for making the augmentedsunscreen-wound healing compositions. In general, the augmentedcompositions are made by admixing the therapeutic sunscreen-woundhealing composition with the medicament useful for treating wounds toprepare the augmented sunscreen-wound healing composition.

The present invention also extends to methods for employing theaugmented sunscreen-wound healing compositions. In general, an augmentedsunscreen-wound healing composition is employed by contacting thecomposition with the skin to be exposed to the sun. In a specificembodiment, the invention is directed to a method for minimizing andtreating sunburn in a human with an augmented sunscreen-wound healingcomposition (I.A+M7+M) which comprises the steps of:

(A) providing a therapeutically effective amount of a sunscreen-woundhealing composition which comprises:

(1) a sunscreen agent;

(2) an anti-inflammatory agent; and

(3) a wound healing composition, wherein the wound healing compositioncomprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a medicament useful for treating wounds.

(C) contacting the augmented sunscreen-wound healing composition withthe skin of the human prior to exposure to the sun.

The types of wounds which may be healed using the sunscreen-woundhealing compositions and the augmented sunscreen-wound healingcompositions of the present invention are inflammation wounds induced bysunburn. The therapeutic compositions may be used topically to protectand accelerate the healing of injured tissue.

Methods for treating sunburn comprise topically administering thecompositions of the present invention directly to the skin of the humanprior to exposure to the sun. The composition is maintained in contactwith the skin for a period of time sufficient to increase theproliferation and resuscitation rate of the cells.

e. Formulations of the Sunscreen-Wound Healing Compositions ofEmbodiment Two (I.A-D+M7) and (I.A-D+M7+M)

Once prepared, the inventive therapeutic sunscreen-wound healingcompositions and augmented sunscreen-wound healing compositions may bestored for future use or may be formulated in effective amounts withpharmaceutically acceptable carriers such as pharmaceutical appliancesand topical vehicles to prepare a wide variety of pharmaceuticalcompositions. The pharmaceutically acceptable carriers which may beemployed and the methods used to prepare the pharmaceutical compositionshave been described above in connection with the formulations of thewound healing compositions of Embodiment One (I.A-D).

In a preferred embodiment, the invention is directed to asunscreen-wound healing pharmaceutical composition which comprises:

(A) a therapeutic sunscreen-wound healing composition (I.A+M7) whichcomprises:

(1) a sunscreen agent;

(2) an anti-inflammatory agent; and

(3) a wound healing composition, wherein the wound healing compositioncomprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a pharmaceutically acceptable carrier selected from the groupconsisting of pharmaceutical appliances, bioadhesives, and occlusivevehicles.

In another preferred embodiment, the invention is directed to a methodfor preparing a pharmaceutical composition for increasing theproliferation and resuscitation rate of mammalian cells, which comprisesthe steps of:

(A) providing a therapeutically effective amount of a sunscreen-woundhealing composition (I.A+M7) which comprises:

(1) a sunscreen agent;

(2) an anti-inflammatory agent; and

(3) a wound healing composition comprising:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(B) providing a pharmaceutically acceptable carrier; and

(C) admixing the sunscreen-wound healing composition from step (A) andthe pharmaceutically acceptable carrier from step (B) to form apharmaceutical composition.

f. Examples of the Sunscreen-Wound Healing Compositions of EmbodimentTwo (I.A-D+M7) Study 1 Summary

This study was conducted to determine the potential of formulations withmoisturizers and antioxidants to effectively prevent or reduce solarsimulator derived erythema in normal volunteers, and whether theerythema once manifested would be ameliorated with the test product. Theconcept was to develop a product that could protect and heal skin fromUV damage without the use of UV sunscreen blockers, i.e., PABA,oxybenzone, etc. Such protection would be extremely beneficial for coldsores and psoriatic lesions since UV exposure is known to exacerbatethese skin diseases.

Two products, a lotion and a cream, were evaluated for their sunprotection potential (Phase I) and for product efficacy in the treatmentof solar simulator induced erythema (Phase II). Ten subjects completedPhase I of the study. Thirteen subjects completed Phase II of the study.An 8% Homosalate solution served as a control in Phase I.

Under the conditions employed in this study, both the cream and lotiontest products provided low level sun protection (SPF values less then 2)and minor improvements in UV-induced erythema as compared withirradiated control sites not treated with test products.

Introduction

Objective

This study was conducted to determine the potential for two woundhealing compositions containing formulations for preventing solarsimulator derived erythema in normal volunteers and whether, theerythema once manifested, would be ameliorated with the test woundhealing composition product.

Rationale

The demands for sun screen and skin care products make up the fastestgrowing cosmetics sector in the United States. The growth in sun careproducts is continuing because overexposure to the sun is believed toproduce many ill-effects on the skin. It is known that even high sunprotectant factor (SPF-15) products do not fully protect human skinagainst cumulative skin damage. The nature of this damage is believed tobe associated with the presence of various "reactive" oxygen species. Aproduct to reduce the levels of photogenerated reactive oxygen speciesshould provide meaningful long term skin benefits. The purpose of thisexperiment was to determine whether a formula which containsmoisturizers and antioxidants could effectively eliminate or reduce thedamage produced from overexposure to UV light. The objective was also todetermine if applying the product before and/or after UV damage wouldprovide beneficial effects to the damaged area.

Background

Two (2) products were submitted for testing in this two phase study.Each study group consisted of ten (10) volunteer subjects. The productswere considered reasonably safe for testing on human subjects. Theprotocol followed for Phase I was that established by the OTC panel andpublished in the Federal Register, Aug. 25, 1978 (Vol 43, No. 166).

Materials and Methods

A sample of each test material(s) was reserved and stored for a periodof five (5) years. At the conclusion of the clinical study, theremaining test material(s) was discarded. All information regarding thereceipt, storage and disposition of the material(s) was also recorded ona Clinical Material Record form. All test materials were kept in alocked product storage room accessible to clinical staff members only.

Test Materials

Product (1) Identification: Lotion

Description: White Lotion

Quantity Provided: 1×6 oz.

Amount applied: 0.1 g/50 cm²

Product (2) Identification: Cream

Description: Yellow Cream

Quantity Provided: 1×4 oz.

Amount applied: 0.1 g/50 cm²

Product (1) was Lubriderm™ Lotion containing 2% sodium pyruvate, 1%vitamin E, and 1% chicken fat. Product (2) was Lubriderm™ Creamcontaining aquaphor/petrolatum cream with 10% sodium pyruvate, 5%vitamin E, and 5% chicken fat.

Experimental Design

Inclusion Data

Individuals included in the study were eighteen (18) years of age orolder; were fair and had uniformly-colored skin on the lower thoracicarea of the back which would allow a discernible erythema; were free ofany systemic or dermatologic disorder which would interfere with thetest results; had completed a phototesting Medical Screening form and aMedical/Personal History form; and had read, understood, and signed aninformed consent agreement.

Exclusion Data

Individuals excluded in the study were individuals who had any visibleskin disease at the test site which would have interfered with the testresults; were receiving systemic or topical drugs or medication such asantihistamines or anti-inflammatories which might have interfered withthe test results; were taking medication suspected of causingphotobiological reactions (i.e., tetracyclins, thiazides, etc.); hadactive atopic dermatitis or eczema; had psoriasis; were currently undertreatment for asthma; had cataracts; had a history of skin cancer; werepregnant or planning a pregnancy or nursing a child; or has a knownsensitivity to cosmetics, skin care products, or topical drugs asrelated to the products being tested.

Light Source

A Xenon Arc Solar Simulator (150 W) was used which has a continuousemission spectrum in the UVA and UVB range (290 to 400 nanometers). Lessthan 1% of its total energy was composed of "non-solar" wavelengthsbelow 290 mm. The output was monitored daily, using the Robert-Bergemeter. Additional measurements of the energy output were taken andrecorded as needed.

Definitions of Terms in Clinical Evaluation

MED, Minimal Erythema Dose

The time of light exposure necessary to produce a minimal perceptibleerythema (redness) on the skin, discernible sixteen (16) to twenty four(24) hours later.

SPF, Sun Protection Factor

The ultraviolet energy required to produce an MED on protected skin,divided by the ultraviolet energy required to produce an MED onunprotected skin.

PCD, Product Category Designation

1. Minimal Sun Protection Product

Sunscreen products that provide an SPF value of 2 to 4, and offer theleast protection from sunburning, but permit suntanning.

2. Moderate Sun Protection Product

Sunscreen products that provide an SPF value of 4 to 6, and offermoderate protection from sunburning, and permit some suntanning.

3. Extra Sun Protection Product

Sunscreen products that provide an SPF value of 6 to 8, and offer extraprotection from sunburning, and permit limited suntanning.

4. Maximal Sun Protection Product

Sunscreen products that provide an SPF value of 8 to under 15, and offermaximal protection from sunburning, and permit little or no suntanning.

5. Ultra Sun Protection Product

Sunscreen products that provide an SPF value of 15 or greater, and offerthe most protection from sunburning and permit no suntanning.

Phase I--Determination of Minimal Erythemal Dose (MED)

MED is defined as the time of light exposure required to produce aminimal perceptible erythema reaction discernible sixteen (16) totwenty-four (24) hours after irradiation using a standardizedultraviolet light source that emits UVB(290-320 nm) as all or part ofits emission spectrum. Subjects who were candidates for this testinghave skin types commonly referred to as Fitzpatrick skin types, ofCategory I, II, or III according to the following definitions:

Category I Always burns easily, never tans.

Category II Always burns easily, tans minimally.

Category III Burns moderately, tans gradually (light brown).

Category IV Burns minimally, always tans well (moderate brown).

Category V Rarely burns, tans very well (moderate brown).

Category VI Never burns, deeply pigmented.

An area, other than the test site, was drawn with a marking pen on thesubject's back and divided into five (5) equal sites. The anticipatedMED was estimated from skin type (I, II, or III) of the individual andthe irradiation size calculated based on energy output of the Xenonlamp. The (5) sites were irradiated on the basis of a geometricprofession of 1.25, i.e., each site receiving 25% more exposure than thesite to its left. Sixteen (16) to twenty-four (24) hours later, the MEDwas determined by establishing the site which exhibited the least amountof perceptible erythema.

SPF Determination

The test material was applied as follows:

A 50 cm² area was marked on the back of the subject. Then, a uniformapplication of 0.1 g or ml or product was applied to the area withcareful spreading and gentle but thorough rubbing over the entire markedarea. The product was permitted to dry for fifteen (15) to thirty (30)minutes.

The MED previously determined on untreated skin was multiplied by theexpected SPF of the product. For example, for an MED of twenty-five (25)seconds, and a test product whose SPF is estimated at two (2), themedian exposure time for this product would be 2×25 seconds=50 seconds.

The irradiation for site exposure was again calculated on the basis of ageometric progression of 1.25 with the site to the right receiving moreirradiation than the site to its left. The sites were then exposed toultraviolet light from the solar simulator. The procedure was repeatedusing the control product, an 8% homosalate standard.

In addition, an area of untreated skin was exposed to ultraviolet lightfrom the solar simulator to again determine the MED of unprotected skin.Sixteen (16) to twenty-four (24) hours later, the MED for the treatedand untreated skin was determined and the SPF calculated.

Following exposure, all immediate responses were noted for reddening,vesiculation, tanning, darkening of skin, etc.

Study Flow Chart

    ______________________________________    Day    ______________________________________    1      Obtained informed consent; completed medical screening form;           conducted UV irradiation for MED determination.    2      Determined MED; calculated UV exposure times;           irradiated test product site, control product site,           and untreated sites for second MED determination.    3      Evaluated all sites; calculated SPF.    ______________________________________

To assure the uniform evaluation of sunscreen products, a standardsunscreen was used concomitantly in the test procedure, specifically instep 2, irradiated control product site. This control product was aneight percent (8%) homosalate preparation.

Rejection of Test Data

There were three (3) reasons for rejection of test data.

1. Sometimes the exposure either failed to elicit an MED response on thetreated or unprotected skin sites or elicited responses at all five (5)irradiated sites. In either event, that test was considered a technicalfailure and had to be discarded. If the subject reacted to one or moreexposures on the unprotected control site, but not on the treated site,then a minimal estimate could be obtained. However, this estimate wouldnot be used in assessing the Mean of the SPF values.

2. The response on the treated sites was randomly absent, whichindicated the product was not spread evenly. Therefore, no assessment ofprotection was possible.

3. If the SPF values obtained experimentally were well outside theexpected range, the values were discarded.

Phase II--Treatment of Erythema

Ten subjects whose MEDs were determined by the previous method (Phase I)had twelve test site areas delineated on the back: five sites wereexposed to one times the MED; five sites were exposed to two times theMED, and two sites were not exposed to UV light. The cream test productwas applied (0.1 g/50 cm²) and rubbed into two of the 1×MED sites, twoof the 2×MED sites, and to one of the unexposed sites. The lotion testproduct was applied to two separate 1×MED sites, two separate 1×MEDsites, and one separate unexposed site. The two remaining exposed areas(one 1×MED and one 2×MED) served as controls and did not have productapplied. All sites were covered, but not occluded, with a gauzedressing. Application of the product to the same sites was repeatedapproximately six hours later and the following mornings and afternoonsfor four consecutive days. Evaluation of the test sites was made eachafternoon prior to product application by a trained clinical evaluatorwho had no participation in any other aspect of the study procedure. Afinal evaluation was made on the morning of Day 8, the last productapplication having occurred on the previous Friday (Day 4). Evaluationsof each test site were made according to the following scale:

0.0--minimal or doubtful erythema

0.5--faint erythema

1.0--distinct, but mild erythema

2.0--moderate erythema

Results and Discussion

Phase I

Two (2) products, a lotion and a cream, were submitted for evaluation oftheir sun protective potential. A total of ten (10) subjects between theages of 25 and 54 were enrolled and completed the evaluation of the testproducts and the homosalate control.

A low level of sun protection was observed; an SPF value of 1.9±0.2(N=9) was obtained for the cream and a value of 1.7±0.3 (N=8) wasobtained for the lotion.

Phase II

The same two (2) products were also evaluated for their product efficacyin the treatment of solar-simulator induced erythema. A total of fifteen(15) subjects between the ages of 20 and 66 were enrolled and thirteen(13) subjects completed the study. Two (2) subjects discontinued forpersonal reasons.

Product treated sites exposed to two MEDs of irradiation exhibited minorimprovements in resolution of erythema as compared to the irradiatednon-product treated sites. Comparisons between erythema levels at Day 2and subsequent evaluation days was used as the basis of analysis. Day 2was selected as the baseline day since increases in erythema from Day 1to Day 2 occurred frequently but appeared to have stabilized by Day 2.Comparison of erythema results on Days 3, 4, 5 and 8 to Day 2 suggest aminor acceleration in the resolution of erythema responses.Specifically, in 12 instances among the 52 comparisons (23%), thirteen(13) subjects with 4 comparisons each, comparing Day 2 to Days 3, 4, 5,and 8, reductions in erythema associated with both the cream and thelotion were manifested.

While it is noted with caution that both the proportion and degree ofimprovement associated with the product test sites were minor, it isalso noteworthy that there was only one instance out of the total of onehundred four (104) comparisons for the two products in which the controlsite showed greater degree of improvement than the test site.Furthermore, this one improvement occurred only for the Day 8/Day 2comparison.

The cream product showed improvement over the control site in twosubjects for all four comparison intervals, in one subject at two of thefour intervals, and two other subjects at one of the four intervals.Similarly, the lotion product showed improvement in one subject at allfour intervals, in one subject at three intervals, in two subjects attwo intervals, and, in one subject at one interval. Conversely, eight ofthirteen subjects showed no differences at any of the four intervals incomparisons between the test product sites and the control sites forboth the cream and the lotion products. While the effects noted heremust again be stressed as minimal, it is, nonetheless, noteworthy thatimprovements, where noted, favored effects of the test products over thecontrol and not the opposite: twenty-four instances of possible positiveeffects from the two test products compared to one such possibleinstance at the control site.

A similar analysis of data associated with the 1×MED sites did notprovide the same results due to the low level and variable erythematousresponses observed. This is believed to be due to the fact that minorperturbations of ultra violet lamp output are likely to be sufficient toresult in sub-erythema and mildly erythema responses even on the samesubject at adjoining sites.

In conclusion, under the conditions employed in this clinical study,both the cream and lotion test products provided low-level sunprotection and indications of minor improvement in UV-induced erythemaas compared with irradiated control sites not treated with testproducts. However, it is clear that these effects are, at best, minorand that verification of efficacy would require a larger proportion ofstudy subjects. Formulation changes (vehicle, level of wound healingcomposition) or product application (e.g., multiple applications orocclusive conditions) may also be worth exploring.

8. Dermatological-Wound Healing Compositions a. Embodiment Two(I.A-D+M8)

Applicant has discovered therapeutic dermatological-wound healingcompositions (I.A-D+M8) useful to minimize and treat diaper dermatitis.The compositions comprise a therapeutically effective amount of abuffering agent and an anti-inflammatory agent (buffering agent andanti-inflammatory agent collectively referred to as M8) and a woundhealing composition of Embodiment One (I.A-D). Preferably, the woundhealing composition (I.A) comprises (a) pyruvate, (b) an antioxidant,and (c) a mixture of saturated and unsaturated fatty acids. Bufferingagents can help prevent diaper dermatitis by neutralizing ammonia but donot heal injured mammalian cells. Anti-inflammatory agents can reduceinflammation (erythema) in a patient but do not promote the woundhealing process. Wound healing compositions can increase theresuscitation rate of injured mammalian cells and the proliferation rateof new mammalian cells to replace dead cells. Applicants have found thatthe combination of a buffering agent, an anti-inflammatory agent, and awound healing composition results in a therapeutic dermatological-woundhealing compositions useful for minimizing and treating diaperdermatitis. The dermatological-wound healing compositions may optionallycontain a therapeutically effective amount of a topical antiseptic tofurther reduce the duration and severity of diaper dermatitis.

The combination of the buffering agent, the anti-inflammatory agent, andthe wound healing compositions of the present invention provides apharmaceutical composition useful for minimizing and treating diaperdermatitis and having an enhanced ability to prevent and reduce injuryto mammalian cells and further increase the resuscitation rate ofinjured mammalian cells. The tissue damage associated with diaperdermatitis is believed to be caused by the production of cellularproduced active oxygen species. Combination of the buffering agent,anti-inflammatory agent, and the wound healing compositions helpssuppress such reactive oxygen-linked tissue injury.

Buffering agents are solute compounds which will form a solution towhich moderate amounts of either a strong acid or base may be addedwithout causing a large change in the pH value of the solution. InBronsted's terminology, a buffering agent contains both a weak acid andits conjugate weak base. Buffering solutions usually contain (a) a weakacid and a salt of the weak acid, (b) a mixture of an acid salt with thenormal salt, or (c) a mixture of two acid salts, for example NaH₂ PO₄and Na₂ HPO₄. A weak acid becomes a buffering agent when alkali is addedand a weak base becomes a buffering agent when acid is added. Thebuffering agents in the dermatological-wound healing compositions of thepresent invention may be selected from a wide range of therapeuticagents and mixtures of therapeutic agents. Buffering agents which occurin nature include phosphates, carbonates, ammonium salts, proteins ofplant and animal tissues, and the carbonic-acid-bicarbonate system inblood. Nonlimiting illustrative specific examples of buffering agentsinclude citric acid-sodium titrate solution, phosphoric acid-sodiumphosphate solution, and acetic acid-sodium acetate solution. Preferably,the buffering agent is phosphoric acid-sodium phosphate.

The amount of buffering agent used in the present invention is aneffective amount and may vary depending upon the dosage recommended orpermitted for the particular buffering agent. In general, the amount ofbuffering agent present is the ordinary dosage required to obtain thedesired result. Such dosages are known to the skilled practitioner inthe medical arts and are not a part of the present invention. In apreferred embodiment, the buffering agent in the dermatological-woundhealing composition is present in an amount to maintain the pH of thedermatitis in a range from about 5 to about 8, preferably from about 5.5to about 7.5, and more preferably from about 6 to about 7.

Anti-inflammatory agents are compounds that counteract or suppress theinflammatory process. The anti-inflammatory agents in thedermatological-wound healing compositions of the present invention maybe selected from a wide variety of steroidal, non-steroidal, andsalicylate water-soluble and water-insoluble drugs and their acidaddition or metallic salts. Both organic and inorganic salts may be usedprovided the anti-inflammatory agent maintains its medicament value. Theanti-inflammatory agents may be selected from a wide range oftherapeutic agents and mixtures of therapeutic agents which may beadministered in sustained release or prolonged action form. Nonlimitingillustrative specific examples of non-steroidal anti-inflammatory agentsinclude the following medicaments: ibuprofen, naproxen, sulindac,diflunisal, piroxicam, indomethacin, etodolac, meclofenamate sodium,fenoproben calcium, ketoprofen, mefenamic acid, nabumetone, ketorolactromethamine, diclofenac, and evening primrose oil (containing about 72%linoleic acid and about 9% gamma-linolenic acid). Nonlimitingillustrative specific examples of salicylate anti-inflammatory agentsinclude the following medicaments: acetylsalicylic acid, mesalamine,salsalate, diflunisal, salicylsalicylic acid, and choline magnesiumtrisalicylate. Nonlimiting illustrative specific examples of steroidalanti-inflammatory agents include the following medicaments: flunisolide,triamcinoline, triamcinoline acetonide, beclomethasone diproprionate,betamethasone diproprionate, hydrocortisone, cortisone, dexamethasone,prednisone, methyl prednisolone, and prednisolone.

Preferred anti-inflammatory agents to be employed may be selected fromthe group consisting of ibuprofen, naproxen, sulindac, diflunisal,piroxicam, indomethacin, etodolac, meclofenamate sodium, fenoprobencalcium, ketoprofen, mefenamic acid, nabumetone, ketorolac tromethamine,diclofenac, evening primrose oil, acetylsalicylic acid, mesalamine,salsalate, diflunisal, salicylsalicylic acid, choline magnesiumtrisalicylate, flunisolide, triamcinoline, triamcinoline acetonide,beclomethasone diproprionate, betamethasone diproprionate,hydrocortisone, cortisone, dexamethasone, prednisone, methylprednisolone, and prednisolone. In a preferred embodiment, theanti-inflammatory agent is selected from the group consisting ofibuprofen, naproxen, sulindac, diflunisal, piroxicam, indomethacin,etodolac, meclofenamate sodium, fenoproben calcium, ketoprofen,mefenamic acid, nabumetone, ketorolac tromethamine, diclofenac, andevening primrose oil. In a more preferred embodiment, theanti-inflammatory agent is evening primrose oil.

The anti-inflammatory agent of the present invention may be used in manydistinct physical forms well known in the pharmaceutical art to providean initial dosage of the anti-inflammatory agent and/or a furthertime-release form of the anti-inflammatory agent. Without being limitedthereto, such physical forms include free forms and encapsulated forms,and mixtures thereof.

The amount of anti-inflammatory agent used in the present invention is atherapeutically effective amount and may vary depending upon thetherapeutic dosage recommended or permitted for the particularanti-inflammatory agent. In general, the amount of anti-inflammatoryagent present is the ordinary dosage required to obtain the desiredresult. Such dosages are known to the skilled practitioner in themedical arts and are not a part of the present invention. In a preferredembodiment, the anti-inflammatory agent in the dermatological-woundhealing composition is present in an amount from about 0.01% to about10%, preferably from about 0.1% to about 5%, and more preferably fromabout 1% to about 3%, by weight.

In yet another preferred embodiment, the therapeuticdermatological-wound healing compositions of the present inventionfurther comprise a topical antiseptic agent. Antiseptic agents arecompounds that inhibit the growth and development of microorganismswithout necessarily destroying the microorganism. The antiseptic agentsin the dermatological-wound healing compositions of the presentinvention may be selected from a wide range of therapeutic agents andmixtures of therapeutic agents. Nonlimiting illustrative specificexamples of topical antiseptic agents include zinc oxide, and calciumundecylenate.

The amount of antiseptic agent used in the present invention is atherapeutically effective amount and may vary depending upon thetherapeutic dosage recommended or permitted for the particularantiseptic agents. In general, the amount of antiseptic agents presentis the ordinary dosage required to obtain the desired result. Suchdosages are known to the skilled practitioner in the medical arts andare not a part of the present invention. In a preferred embodiment, theantiseptic agent in the dermatological-wound healing composition ispresent in an amount from about 1% to about 30%, preferably from about2% to about 25%, and more preferably from about 5% to about 20%, byweight.

b. Methods for Making the Dermatological-Wound Healing Compositions ofEmbodiment Two (I.A-D+M8)

The present invention extends to methods for making the therapeuticdermatological-wound healing compositions (I.A-D+M8). In general, atherapeutic dermatological-wound healing composition is made by formingan admixture of the wound healing components of Embodiment One (I.A-D),a buffering agent, an anti-inflammatory agent. In a first aspect ofEmbodiment Two (I.A+M8), a dermatological-wound healing therapeuticcomposition is made by forming an admixture of a buffering agent, ananti-inflammatory agent, and a wound healing composition comprising (a)a pyruvate, (b) an antioxidant, and (c) a mixture of saturated andunsaturated fatty adds. In a second aspect of Embodiment Two (I.B+M8), adermatological-wound healing therapeutic composition is made by formingan admixture of a buffering agent, an anti-inflammatory agent, and awound healing composition comprising (a) a pyruvate, (b) a lactate, and(c) a mixture of saturated and unsaturated fatty acids. In a thirdaspect of Embodiment Two (I.C+M8), a dermatological-wound healingtherapeutic composition is made by forming an admixture of a bufferingagent, an anti-inflammatory agent, and a wound healing compositioncomprising (a) an antioxidant, and (b) a mixture of saturated andunsaturated fatty acids. In a fourth aspect of Embodiment Two (I.D+M8),a dermatological-wound healing therapeutic composition is made byforming an admixture of admixture of a buffering agent, ananti-inflammatory agent, and a wound healing composition comprising (a)a lactate, (b) an antioxidant, and (c) a mixture of saturated andunsaturated fatty acids.

In a preferred embodiment, the invention is directed to a method forpreparing a therapeutic dermatological-wound healing composition(I.A+M8) useful to minimize and treat diaper dermatitis which comprisesthe steps of admixing a therapeutically effective amount of thefollowing ingredients:

(1) a buffering agent to maintain the pH of the dermatitis in a rangefrom about 5 to about 8;

(2) an anti-inflammatory agent; and

(3) a wound healing composition comprising:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

c. Methods for Employing the Dermatological-Wound Healing Compositionsof Embodiment Two (I.A-D+M8)

The present invention extends to methods for employing the therapeuticdermatological-wound healing compositions (I.A-D+M8). In general, atherapeutic composition is employed by contacting the therapeuticcomposition with the diaper dermitis. In a preferred embodiment, theinvention is directed to a method for minimizing and treating diaperdermatitis in a human which comprises the steps of (I.A+M8):

(A) providing a therapeutically effective amount of adermatological-wound healing composition which comprises:

(1) a buffering agent to maintain the pH of the dermatitis in a rangefrom about 5 to about 8;

(2) an anti-inflammatory agent; and

(3) a wound healing composition comprising:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) contacting the dermatological-wound healing composition with thedermatitis.

d. Augmented Dermatological-Wound Healing Compositions of Embodiment Two(I.A-D+M8+M)

In another aspect of Embodiment Two, the therapeuticdermatological-wound healing compositions (I.A-D+M8) of the presentinvention may be further combined with medicaments useful for treatingwounds (M) to form augmented dermatological-wound healing compositions(I.A-D+M8+M). In this embodiment, the combination of thedermatological-wound healing composition of the present invention andthe medicament useful for treating wounds provides an augmenteddermatological-wound healing composition having an enhanced ability toincrease the proliferation and resuscitation rate of mammalian cells.For example, the therapeutic compositions of the present invention maybe used in combination with medicaments useful for treating wounds suchas immunostimulating agents (Betafectin™), antiviral agents,antikeratolytic agents, anti-inflammatory agents, antifungal agents,tretinoin, sunscreen agents, other dermatological agents, topicalantihistamine agents, antibacterial agents, bioadhesive agents,respiratory bursting inhibitors (lactic acid, adenosine), inhibitors ofprostaglandin synthesis (ibuprofen, aspirin, indomethacin, meclofenomicacid, retinoic acid, padimate O, meclomen, oxybenzone), steroidalanti-inflammatory agents (corticosteroids including synthetic analogs),antimicrobial agents (neosporin ointment, silvadine), antiseptic agents,anesthetic agents (pramoxine hydrochloride, lidocaine, benzocaine), cellnutrient media, burn relief medications, sun burn medications, acnepreparations, insect bite and sting medications, wound cleansers, wounddressings, scar reducing agents (vitamin E), and the like, and mixturesthereof, to further enhance the proliferation and resuscitation rate ofmammalian cells. Preferably, the medicament useful for treating woundsis selected from the group consisting of immunostimulating agents,antiviral agents, antikeratolytic agents, anti-inflammatory agents,antifungal agents, tretinoin, sunscreen agents, dermatological agents,topical antihistamine agents, antibacterial agents, bioadhesive agents,respiratory bursting inhibitors, inhibitors of prostaglandin synthesis,antimicrobial agents, cell nutrient media, scar reducing agents, andmixtures thereof. More preferably, the medicament useful for treatingwounds is selected from the group consisting of immunostimulatingagents, antiviral agents, antikeratolytic agents, anti-inflammatoryagents, antifungal agents, acne treating agents, sunscreen agents,dermatological agents, antihistamine agents, antibacterial agents,bioadhesive agents, and mixtures thereof.

In a preferred embodiment, the invention is directed to an augmenteddermatological-wound healing composition (I.A+M8+M) useful to minimizeand treat diaper dermatitis which comprises:

(A) a therapeutic dermatological-wound healing composition whichcomprises a therapeutically effective amount of:

(1) a buffering agent to maintain the pH of the dermatitis in a rangefrom about 5 to about 8;

(2) an anti-inflammatory agent; and

(3) a wound healing composition comprising:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic add, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a medicament useful for treating wounds.

The present invention extends to methods for making the augmenteddermatological-wound healing compositions. In general, the augmentedcompositions are made by admixing the therapeutic dermatological-woundhealing composition with the medicament useful for treating wounds toprepare the augmented dermatological-wound healing composition.

The present invention also extends to methods for employing theaugmented dermatological-wound healing compositions. In general, anaugmented dermatological-wound healing composition is employed bycontacting the composition with the dermitis. In a preferred embodiment,the invention is directed to a method for minimizing and treating diaperdermatitis in a human with an augmented dermatological-wound healingcomposition (I.A+M8+M) which comprises the steps of:

(A) providing a therapeutically effective amount of adermatological-wound healing composition which comprises:

(1) a buffering agent to maintain the pH of the dermatitis in a rangefrom about 5 to about 8;

(2) an anti-inflammatory agent; and

(3) a wound healing composition comprising:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a medicament useful for treating wounds; and

(C) contacting the augmented dermatological-wound healing compositionwith the dermatitis.

The types of wounds which may be healed using the dermatological-woundhealing compositions and the augmented dermatological-wound healingcompositions of the present invention are wounds induced by diaperdermatitis. The therapeutic compositions may be used topically toprotect and accelerate the healing of injured tissue.

Methods for treating diaper dermatitis comprise topically administeringthe compositions of the present invention directly to the dermatitis.The composition is maintained in contact with the skin for a period oftime sufficient to increase the proliferation and resuscitation rate ofthe cells.

e. Formulations of the Dermatological-Wound Healing Compositions ofEmbodiment Two (I.A-D+M8) and (I.A-D+M8+M)

Once prepared, the inventive therapeutic dermatological-wound healingcompositions and augmented dermatological-wound healing compositions maybe stored for future use or may be formulated in effective amounts withpharmaceutically acceptable carriers such as pharmaceutical appliancesand topical vehicles to prepare a wide variety of pharmaceuticalcompositions. The pharmaceutically acceptable carriers which may beemployed and the methods used to prepare the pharmaceutical compositionshave been described above in connection with the formulations of thewound healing compositions of Embodiment One (I.A-D).

In a preferred embodiment, the invention is directed to adermatological-wound healing pharmaceutical composition which comprises:

(A) a therapeutic dermatological-wound healing composition (I.A+M8)which comprises:

(1) a buffering agent to maintain the pH of the dermatitis in a rangefrom about 5 to about 8;

(2) an anti-inflammatory agent; and

(3) a wound healing composition comprising:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a pharmaceutically acceptable carrier selected from the groupconsisting of pharmaceutical appliances, bioadhesives, and occlusivevehicles.

In another preferred embodiment, the invention is directed to a methodfor preparing a pharmaceutical composition for increasing theproliferation and resuscitation rate of mammalian cells, which comprisesthe steps of:

(A) providing a therapeutically effective amount of adermatological-wound healing composition (I.A+M8) which comprises:

(1) a buffering agent;

(2) an anti-inflammatory agent; and

(3) a wound healing composition comprising:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(B) providing a pharmaceutically acceptable carrier; and

(C) admixing the dermatological-wound healing composition from step (A)and the pharmaceutically acceptable carrier from step (B) to form apharmaceutical composition.

9. Antihistamine-Wound Healing Compositions a. Embodiment Two (I.A-D+M9)

Applicant has discovered therapeutic antihistamine-wound healingcompositions (I.A-D+M9) which comprise a topical antihistamine agent(M9) and the wound healing compositions of Embodiment One (I.A-D).Preferably, the wound healing composition (I.A) comprises (a) pyruvate,(b) an antioxidant, and (c) a mixture of saturated and unsaturated fattyacids. Topical antihistamine agents are useful for the topical treatmentof skin irritations but are potentially sensitizing. Wound healingcompositions can increase the resuscitation rate of injured mammaliancells and the proliferation rate of new mammalian cells to replace deadcells but do not treat itching. The combination of a topicalantihistamine agent and a wound healing composition results in atherapeutic antihistamine-wound healing composition which reduces theduration and severity of itching associated with skin irritations(augmented wound healing composition) and the sensitizing effectassociated with topical antihistamine agents (cytoprotective-woundhealing composition).

The combination of a topical antihistamine agent and the wound healingcompositions of the present invention provides a pharmaceuticalcomposition useful for treating itching associated with skin irritationsand having an enhanced ability to prevent and reduce injury to mammaliancells and further increase the resuscitation rate of injured mammaliancells. The tissue damage associated with a topical antihistamine agentinduced sensitivity is believed to be caused by the production ofcellular produced active oxygen species. Combination of a topicalantihistamine agent and the wound healing compositions may suppress suchreactive oxygen-linked tissue injury.

Topical antihistamine agents are compounds that counter the action ofhistamine. Histamine causes dilation of capillaries, which increasescapillary permeability and results in a drop in blood pressure,constriction of the bronchial smooth muscle of the lung, and inductionof increase gastric secretion. Topical antihistamine agents areimplicated as mediators of immediate hypersensitivity. The topicalantihistamine agents in the antihistamine-wound healing compositions ofthe present invention may be selected from a wide range of therapeuticagents and mixtures of therapeutic agents. Nonlimiting illustrativespecific examples of topical antihistamine agents includediphenhydramine hydrochloride and pramoxine hydrochloride. Preferably,the topical antihistamine agent is diphenhydramine hydrochloride.

The amount of topical antihistamine agent used in the present inventionis a therapeutically effective amount and may vary depending upon thetherapeutic dosage recommended or permitted for the particular topicalantihistamine agent. In general, the amount of topical antihistamineagent present is the ordinary dosage required to obtain the desiredresult. Such dosages are known to the skilled practitioner in themedical arts and are not a part of the present invention. In a preferredembodiment, the topical antihistamine agent in the antihistamine-woundhealing composition is present in an amount from about 0.1% to about10%, preferably from about 1% to about 7%, and more preferably fromabout 1% to about 2%, by weight.

b. Methods for Making the Antihistamine-Wound Healing Compositions ofEmbodiment Two (I.A-D+M9)

The present invention extends to methods for making the therapeuticantihistamine-wound healing compositions (I.A-D+M9). In general, atherapeutic antihistamine-wound healing composition is made by formingan admixture of the wound healing components of Embodiment One (I.A-D)and a topical antihistamine agent. In a first aspect of Embodiment Two(I.A+M9), an antihistamine-wound healing therapeutic composition is madeby forming an admixture of a topical antihistamine agent and a woundhealing composition comprising (a) a pyruvate, (b) an antioxidant, and(c) a mixture of saturated and unsaturated fatty acids. In a secondaspect of Embodiment Two (I.B+M9), an antihistamine-wound healingtherapeutic composition is made by forming an admixture of a topicalantihistamine agent and a wound healing composition comprising (a) apyruvate, (b) a lactate, and (c) a mixture of saturated and unsaturatedfatty acids. In a third aspect of Embodiment Two (I.C+M9), anantihistamine-wound healing therapeutic composition is made by formingan admixture of a topical antihistamine agent and a wound healingcomposition comprising (a) an antioxidant, and (b) a mixture ofsaturated and unsaturated fatty acids. In a fourth aspect of EmbodimentTwo (I.D+M9), an antihistamine-wound healing therapeutic composition ismade by forming an admixture of a topical antihistamine agent and awound healing composition comprising (a) a lactate, (b) an antioxidant,and (c) a mixture of saturated and unsaturated fatty acids.

In a preferred embodiment, the invention is directed to a method forpreparing a therapeutic antihistamine-wound healing composition (I.A+M9)which comprises the steps of admixing the following ingredients:

(A) a therapeutically effective amount of a topical antihistamine agent;and

(B) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

c. Methods for Employing the Antihistamine-Wound Healing Compositions ofEmbodiment Two (I.A-D+M9)

The present invention extends to methods for employing the therapeuticantihistamine-wound healing compositions (I.A-D+M9). In general, atherapeutic composition is employed by contacting the therapeuticcomposition with the itching associated with skin irritations. In apreferred embodiment, the invention is directed to a method for treatingan itching wound in a mammal with an antihistamine-wound healingcomposition (I.A+M9) which comprises the steps of:

(A) providing a therapeutic antihistamine-wound healing compositionwhich comprises:

(1) a therapeutically effective amount of a topical antihistamine agent;and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) contacting the antihistamine-wound healing composition with theitching wound.

Methods for treating itching associated with skin irritations comprisetopically administering the compositions of the present inventiondirectly to the itching associated with skin irritations. Thecomposition is maintained in contact with the itching associated withskin irritations for a period of time sufficient to increase theproliferation and resuscitation rate of the cells.

d. Augmented Antihistamine-Wound Healing Compositions of Embodiment Two(I.A-D+M9+M)

In another aspect of Embodiment Two, the therapeutic antihistamine-woundhealing compositions (I.A-D+M9) of the present invention may be furthercombined with medicaments useful for treating wounds (M) to formaugmented antihistamine-wound healing compositions (I.A-D+M9+M). In thisembodiment, the combination of the antihistamine-would healingcomposition of the present invention and the medicament useful fortreating wounds provides an augmented antihistamine-wound healingcomposition having an enhanced ability to increase the proliferation andresuscitation rate of mammalian cells. For example, the therapeuticcompositions of the present invention may be used in combination withmedicaments useful for treating wounds such as immunostimulating agents(Betafectin™), antiviral agents, antikeratolytic agents,anti-inflammatory agents, antifungal agents, tretinoin, sunscreenagents, dermatological agents, other topical antihistamine agents,antibacterial agents, bioadhesive agents, respiratory burstinginhibitors (lactic acid, adenosine), inhibitors of prostaglandinsynthesis (ibuprofen, aspirin, indomethacin, meclofenomic acid, retinoicacid, padimate O, meclomen, oxybenzone), steroidal anti-inflammatoryagents (corticosteroids including synthetic analogs), antimicrobialagents (neosporin ointment, silvadine), antiseptic agents, anestheticagents (pramoxine hydrochloride, lidocaine, benzocaine), cell nutrientmedia, burn relief medications, sun burn medications, acne preparations,insect bite and sting medications, wound cleansers, wound dressings,scar reducing agents (vitamin E), and the like, and mixtures thereof, tofurther enhance the proliferation and resuscitation rate of mammaliancells. Preferably, the medicament useful for treating wounds is selectedfrom the group consisting of immunostimulating agents, antiviral agents,antikeratolytic agents, anti-inflammatory agents, antifungal agents,tretinoin, sunscreen agents, dermatological agents, topicalantihistamine agents, antibacterial agents, bioadhesive agents,respiratory bursting inhibitors, inhibitors of prostaglandin synthesis,antimicrobial agents, cell nutrient media, scar reducing agents, andmixtures thereof. More preferably, the medicament useful for treatingwounds is selected from the group consisting of immunostimulatingagents, antiviral agents, antikeratolytic agents, anti-inflammatoryagents, antifungal agents, acne treating agents, sunscreen agents,dermatological agents, antihistamine agents, antibacterial agents,bioadhesive agents, and mixtures thereof.

In a preferred embodiment, the invention is directed to an augmentedantihistamine-wound healing composition (I.A+M9+M) which comprises:

(A) a therapeutic antihistamine-wound healing composition whichcomprises:

(1) a therapeutically effective amount of a topical antihistamine agent;and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a medicament useful for treating wounds.

The present invention extends to methods for making the augmentedantihistamine-wound healing compositions. In general, the augmentedcompositions are made by admixing the therapeutic antihistamine-wouldhealing composition with the medicament useful for treating wounds toprepare the augmented antihistamine-wound healing composition.

The present invention also extends to methods for employing theaugmented antihistamine-wound healing compositions. In general, anaugmented antihistamine-wound healing composition is employed bycontacting the composition with the itching associated with skinirritations. In a preferred embodiment, the invention is directed to amethod for treating an itching wound in a mammal with an augmentedantihistamine-wound healing composition (I.A+M9+M) which comprises thesteps of:

(A) providing a therapeutic augmented antihistamine-wound healingcomposition which comprises:

(1) a therapeutically effective amount of a topical antihistamine agent;

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(3) providing a medicament useful for treating wounds; and

(B) contacting the augmented antihistamine-wound healing compositionwith the itching wound.

The types of wounds which may be healed using the antihistamine-woundhealing compositions and the augmented antihistamine-wound healingcompositions of the present invention are those which result from anitching injury which causes epidermal damage.

Methods for healing a wound comprise topically administering thecompositions of the present invention directly to a wound site toincrease the healing rate of the wound. The composition is maintained incontact with the wound for a period of time sufficient to increase theproliferation and resuscitation rate of the cells.

e. Formulations of the Antihistamine-Wound Healing Compositions ofEmbodiment Two (I.A-D+M9) and (I.A-D+M9+M)

Once prepared, the inventive therapeutic antihistamine-wound healingcompositions and augmented antihistamine-wound healing compositions maybe stored for future use or may be formulated in effective amounts withpharmaceutically acceptable carriers such as pharmaceutical appliancesand topical vehicles (oral and non-oral) to prepare a wide variety ofpharmaceutical compositions. The pharmaceutically acceptable carrierswhich may be employed and the methods used to prepare the pharmaceuticalcompositions have been described above in connection with theformulations of the wound healing compositions of Embodiment One(I.A-D).

In a preferred embodiment, the invention is directed to anantihistamine-wound healing pharmaceutical composition which comprises:

(A) a therapeutic antihistamine-wound healing composition (I.A+M9) whichcomprises:

(1) a therapeutically effective amount of a topical antihistamine agent;and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a pharmaceutically acceptable carrier selected from the groupconsisting of pharmaceutical appliances, bioadhesives, and occlusivevehicles.

In another preferred embodiment, the invention is directed to a methodfor preparing a pharmaceutical composition for increasing theproliferation and resuscitation rate of mammalian cells, which comprisesthe steps of:

(A) providing a therapeutically effective amount of anantihistamine-wound healing composition (I.A+M9) which comprises:

(1) a topical antihistamine agent; and

(2) a wound healing composition comprising:

(a) pyruvate Selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(B) providing a pharmaceutically acceptable carrier; and

(C) admixing the antihistamine-wound healing composition from step (A)and the pharmaceutically acceptable carrier from step (B) to form apharmaceutical composition.

10. Antibacterial-Wound Healing Compositions a. Embodiment Two(I.A-D+M10)

Applicant has discovered therapeutic antibacterial-wound healingcompositions (I.A-D+M10) which comprise an antibacterial agent (M10) andthe wound healing compositions of Embodiment One (I.A-D). Preferably,the wound healing composition (I.A) comprises (a) pyruvate, (b) anantioxidant, and (c) a mixture of saturated and unsaturated fatty acids.Antibacterial agents can treat bacterial infections in a patient but donot promote the wound healing process. Wound healing compositions canincrease the resuscitation rate of injured mammalian cells and theproliferation rate of new mammalian cells to replace dead cells but donot reduce virus liters. Applicants have found that the combination ofan antibacterial agent and a wound healing composition results in atherapeutic antibacterial-wound healing composition which reduces thesize, duration, and severity of infected wounds.

The combination of the antibacterial agent and the wound healingcompositions of the present invention provides a pharmaceuticalcomposition useful for treating infected wounds and having an enhancedability to prevent and reduce injury to mammalian cells and furtherincrease the resuscitation rate of injured mammalian cells. The tissuedamage associated with many bacterial diseases is believed to be causedby the production of cellular produced active oxygen species.Combination of the antibacterial agent and the wound healingcompositions may suppress such reactive oxygen-linked tissue injury.

The antibacterial agents which may be employed in theantibacterial-wound healing therapeutic compositions may be selectedfrom a wide variety of water-soluble and water-insoluble drugs and theiracid addition or metallic salts. Both organic and inorganic salts may beused provided the antibacterial agent maintains its medicament value.The antibacterial agents may be selected from a wide range oftherapeutic agents and mixtures of therapeutic agents which may beadministered in sustained release or prolonged action form. Nonlimitingillustrative specific examples of antibacterial agents include bismuthcontaining compounds, sulfonamides; nitrofurans, metronidazole,tinidazole, nimorazole, benzoic acid; aminoglycosides, macrolides,penicillins, polypeptides, tetracyclines, cephalosporins,chloramphenicol, and clidamycin. Preferably, the antibacterial agent isselected from the group consisting of bismuth containing compounds, suchas, without limitation, bismuth aluminate, bismuth subcitrate, bismuthsubgalate, bismuth subsalicylate, and mixtures thereof; thesulfonamides; the nitrofurans, such as nitrofurazone, nitrofurantoin,and furozolidone; and miscellaneous antibacterials such asmetronidazole, tinidazole, nimorazole, and benzoic acid; andantibiotics, including the aminoglycosides, such as gentamycin,neomycin, kanamycin, and streptomycin; the macrolides, such aserythromycin, clindamycin, and rifamycin; the penicillins, such aspenicillin G, penicillin V, Ampicillin and amoxicillin; thepolypeptides, such as bacitracin and polymyxin; the tetracyclines, suchas tetracycline, chlorotetracycline, oxytetracycline, and doxycycline;the cephalosporins, such as cephalexin and cephalothin; andmiscellaneous antibiotics, such as chloramphenicol, and clidamycin. Morepreferably, the antibacterial agent is selected from the groupconsisting of bismuth aluminate, bismuth subcitrate, bismuth subgalate,bismuth subsalicylate, sulfonamides, nitrofurazone, nitrofurantoin,furozolidone, metronidazole, tinidazole, nimorazole, benzoic acid,gentamycin, neomycin, kanamycin, streptomycin, erythromycin,clindamycin, rifamycin, penicillin G, penicillin V, Ampicillinamoxicillin, bacitracin, polymyxin, tetracycline, chlorotetracycline,oxytetracycline, doxycycline, cephalexin, cephalothin, chloramphenicol,and clidamycin.

The antibacterial agent of the present invention may be used in manydistinct physical forms well known in the pharmaceutical art to providean initial dosage of the antibacterial agent and/or a furthertime-release form of the antibacterial agent. Without being limitedthereto, such physical forms include free forms and encapsulated forms,and mixtures thereof.

The amount of antibacterial agent which may be employed in theantibacterial-wound healing therapeutic compositions of the presentinvention may vary depending upon the therapeutic dosage recommended orpermitted for the particular antibacterial agent. In general, the amountof antibacterial agent present is the ordinary dosage required to obtainthe desired result. Such dosages are known to the skilled practitionerin the medical arts and are not a part of the present invention. In apreferred embodiment, the antibacterial agent in the antibacterial-woundhealing composition is present in an amount from about 0.01% to about10%, preferably from about 0.1% to about 5%, and more preferably fromabout 1% to about 3%, by weight.

b. Methods for Making the Antibacterial-Wound Healing Compositions ofEmbodiment Two (I.A-D+M10)

The present invention extends to methods for making the therapeuticantibacterial-wound healing compositions (I.A-D+M10). In general, atherapeutic antibacterial-wound healing composition is made by formingan admixture of the wound healing components of Embodiment One (I.A-D)and an antibacterial agent. In a first aspect of Embodiment Two(I.A+M10), an antibacterial-wound healing therapeutic composition ismade by forming an admixture of an antibacterial agent and a woundhealing composition comprising (a) a pyruvate, (b) an antioxidant, and(c) a mixture of saturated and unsaturated fatty acids. In a secondaspect of Embodiment Two (I.B+M10), an antibacterial-wound healingtherapeutic composition is made by forming an admixture of anantibacterial agent and a wound healing composition comprising (a) apyruvate, (b) a lactate, and (c) a mixture of saturated and unsaturatedfatty acids. In a third aspect of Embodiment Two (I.C+M10), anantibacterial-wound healing therapeutic composition is made by formingan admixture of an antibacterial agent and a wound healing compositioncomprising (a) an antioxidant, and (b) a mixture of saturated andunsaturated fatty acids. In a fourth aspect of Embodiment Two (I.D+M10),an antibacterial-wound healing therapeutic composition is made byforming an admixture of an antibacterial agent and a wound healingcomposition comprising (a) a lactate, (b) an antioxidant, and (c) amixture of saturated and unsaturated fatty acids.

In a preferred embodiment, the invention is directed to a method forpreparing a therapeutic antibacterial-wound healing composition(I.A+M10) which comprises the steps of admixing the followingingredients:

(A) a therapeutically effective amount of an antibacterial agent; and

(B) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

c. Methods for Employing the Antibacterial-Wound Healing Compositions ofEmbodiment Two (I.A-D+M10)

The present invention extends to methods for employing the therapeuticantibacterial-wound healing compositions (I.A-D+M10). In general, atherapeutic composition is employed by contacting the therapeuticcomposition with a wound. In a preferred embodiment, the invention isdirected to a method for healing an infected wound in a mammal with anantibacterial-wound healing composition (I.A+M10) which comprises thesteps of:

(A) providing a therapeutic antibacterial-wound healing compositionwhich comprises:

(1) a therapeutically effective amount of an antibacterial agent; and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) contacting the antibacterial-wound healing composition with theinfected wound.

d. Augmented Antibacterial-Wound Healing Compositions of Embodiment Two(I.A-D+M10+M)

In another aspect of Embodiment Two, the therapeutic antibacterial-woundhealing compositions (I.A-D+M10) of the present invention may be furthercombined with medicaments useful for treating wounds (M) to formaugmented antibacterial-wound healing compositions (I.A-D+M10+M). Inthis embodiment, the combination of the antibacterial-wound healingcomposition of the present invention and the medicament useful fortreating wounds provides an augmented antibacterial-wound healingcomposition having an enhanced ability to increase the proliferation andresuscitation rate of mammalian cells. For example, the therapeuticcompositions of the present invention may be used in combination withmedicaments useful for treating wounds such as immunostimulating agents(Betafectin™), antiviral agents, antikeratolytic agents,anti-inflammatory agents, antifungal agents, tretinoin, sunscreenagents, dermatological agents, topical antihistamine agents, otherantibacterial agents, bioadhesive agents, respiratory burstinginhibitors (lactic acid, adenosine), inhibitors of prostaglandinsynthesis (ibuprofen, aspirin, indomethacin, meclofenomic acid, retinoicacid, padimate O, meclomen, oxybenzone), steroidal anti-inflammatoryagents (corticosteroids including synthetic analogs), antimicrobialagents (neosporin ointment, silvadine), antiseptic agents, anestheticagents (pramoxine hydrochloride, lidocaine, benzocaine), cell nutrientmedia, burn relief medications, sun burn medications, acne preparations,insect bite and sting medications, wound cleansers, wound dressings,scar reducing agents (vitamin E), and the like, and mixtures thereof, tofurther enhance the proliferation and resuscitation rate of mammaliancells. Preferably, the medicament useful for treating wounds is selectedfrom the group consisting of immunostimulating agents, antiviral agents,antikeratolytic agents, anti-inflammatory agents, antifungal agents,tretinoin, sunscreen agents, dermatological agents, topicalantihistamine agents, antibacterial agents, bioadhesive agents,respiratory bursting inhibitors, inhibitors of prostaglandin synthesis,antimicrobial agents, cell nutrient media, scar reducing agents, andmixtures thereof. More preferably, the medicament useful for treatingwounds is selected from the group consisting of immunostimulatingagents, antiviral agents, antikeratolytic agents, anti-inflammatoryagents, antifungal agents, acne treating agents, sunscreen agents,dermatological agents, antihistamine agents, antibacterial agents,bioadhesive agents, and mixtures thereof.

In a preferred embodiment, the invention is directed to an augmentedantibacterial-wound healing composition (I.A+M10+M) which comprises:

(A) a therapeutic antibacterial-wound healing composition whichcomprises:

(1) a therapeutically effective amount of an antibacterial agent; and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a medicament useful for treating wounds.

The present invention extends to methods for making the augmentedantibacterial-wound healing compositions. In general, the augmentedcompositions are made by admixing the therapeutic antibacterial-woundhealing composition with the medicament useful for treating wounds toprepare the augmented antibacterial-wound healing composition.

The present invention also extends to methods for employing theaugmented antibacterial-wound healing compositions. In general, anaugmented antibacterial-wound healing composition is employed bycontacting the composition with a wound. In a preferred embodiment, theinvention is directed to a method for healing an infected wound in amammal with an augmented antibacterial-wound healing composition(I.A+M10+M) which comprises the steps of:

(A) providing a therapeutic augmented antibacterial-wound healingcomposition which comprises:

(1) a therapeutically effective amount of an antibacterial agent;

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(3) providing a medicament useful for treating wounds; and

(B) contacting the augmented antibacterial-wound healing compositionwith the infected wound.

The types of wounds which may be healed using the antibacterial-woundhealing compositions and the augmented antibacterial-wound healingcompositions of the present invention are those which result from aninfected injury which causes epidermal damage. The topical therapeuticcompositions may be used orally in the form of a mouth wash or spray toprotect and accelerate the healing of injured oral tissue.

Methods for healing a wound comprise topically administering thecompositions of the present invention directly to a wound site toincrease the healing rate of the wound. The composition is maintained incontact with the wound for a period of time sufficient to increase theproliferation and resuscitation rate of the cells.

e. Formulations of the Antibacterial-Wound Healing Compositions ofEmbodiment Two (I.A-D+M10) and (I.D+M10+M)

Once prepared, the inventive therapeutic antibacterial-wound healingcompositions and augmented antibacterial-wound healing compositions maybe stored for future use or may be formulated in effective amounts withpharmaceutically acceptable carriers such as pharmaceutical appliancesand topical vehicles (oral and non-oral) to prepare a wide variety ofpharmaceutical compositions. The pharmaceutically acceptable carrierswhich may be employed and the methods used to prepare the pharmaceuticalcompositions have been described above in connection with theformulations of the wound healing compositions of Embodiment One(I.A-D).

In a preferred embodiment, the invention is directed to anantibacterial-wound healing pharmaceutical composition which comprises:

(A) a therapeutic antibacterial-wound healing composition (I.A+M10)which comprises:

(1) a therapeutically effective amount of an antibacterial agent; and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a pharmaceutically acceptable carrier selected from the groupconsisting of pharmaceutical appliances, bioadhesives, and occlusivevehicles.

In another preferred embodiment, the invention is directed to a methodfor preparing a pharmaceutical composition for increasing theproliferation and resuscitation rate of mammalian cells, which comprisesthe steps of:

(A) providing a therapeutically effective amount of anantibacterial-wound healing composition (I.A+M10) which comprises:

(1) an antibacterial agent; and

(2) a wound healing composition comprising:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(B) providing a pharmaceutically acceptable carrier; and

(C) admixing the antibacterial-wound healing composition from step (A)and the pharmaceutically acceptable carrier from step (B) to form apharmaceutical composition.

11. Bioadhesive-Wound Healing Compositions A. Embodiment Two (I.A-D+M11)

Applicant has discovered therapeutic bioadhesive-wound healingcompositions (I.A-D+M11) which comprise a bioadhesive agent (M11) andthe wound healing compositions of Embodiment One (I.A-D). Preferably,the wound healing composition (I.A) comprises (a) pyruvate, (b) anantioxidant, and (c) a mixture of saturated and unsaturated fatty acids.The bioadhesive agent comprises a water-swellable but water-insolublefibrous cross-linked material which adheres to live or freshly killedmucous membranes or skin tissues. Applicants have found that thecombination of a bioadhesive agent and a wound healing compositionresults in a therapeutic bioadhesive-wound healing composition which canincrease the resuscitation rate of injured mammalian cells and theproliferation rate of new mammalian cells to replace dead cells andthereby reduce the duration and severity of wounds. Thebioadhesive-wound healing therapeutic compositions may further comprisemedicaments such as immunostimulating agents, cytotoxic agents,antiviral agents, antikeratolytic agents, anti-inflammatory agents,antifungal agents, tretinoin, sunscreen agents, dermatological agents,topical antihistamine agents, antibacterial agents, and the like.

Gingivitis is caused by supragingival plaque which releases toxins andmicrobial products that attack the gingiva and result in inflammation ofthe gingival tissues. Inflammation in the connective tissue results inpocket formation and may ultimately result in periodontitis. The woundhealing compositions of the present invention and other medicaments suchas immunostimulating agents, antiviral agents, antikeratolytic agents,anti-inflammatory agents, antifungal agents, topical antihistamineagents, antibacterial agents, and the like, may help decreaseinflammation and improve healing of the damaged tissues. The use ofsuitable bioadhesive agents may improve coating, prolong duration ofaction, and improve patient's comfort and compliance. Suitable dosageforms for the bioadhesive-wound healing therapeutic compositions aresolution, suspension, gel, paste, dental floss, microcapsules,nanoparticles, liposomes, mono- and multilaminated strips and patches,mono- and multilayered tablets and other suitable bioadhesive deliverydosage forms. The formulation may be delivered non-specifically to thebuccal cavity or locally to the gingiva, gum, gingival pockets, betweenteeth or other suitable target areas.

The combination of a bioadhesive agent and the wound healingcompositions of the present invention provides a bioadhesive agent-woundhealing composition useful for treating wounds and having an enhancedability to prevent and reduce injury to mammalian cells and furtherincrease the resuscitation rate of injured mammalian cells. The tissuedamage associated with wounds is believed to be caused by the productionof cellular produced active oxygen species. Combination of a bioadhesiveagent and the wound healing compositions may suppress such reactiveoxygen-linked tissue injury.

The bioadhesive agents which may be employed in the bioadhesiveagent-wound healing composition of the present invention are materialswhich adhere to live or freshly killed mucous membranes or skin tissues.The bioadhesive agents may be selected from a wide variety ofwater-swellable but water-insoluble fibrous cross-linked materials.Bioadhesives generally comprise a mucoadhesive hydrogel such as apolyacrylic acid cross linked by a polyhydroxy compound such as acarbohydrate (sugar, cyclitols) to form a substantially water-insolublehydrogel. Other bioadhesives include carboxymethylcellulose (CMC),methylcellulose, guar gum, and polycarbophils which are high molecularweight polymers of acrylic acid such as Carbopol™ commercially availablefrom BF Goodrich Company, Cleveland, Ohio. Bioadhesives are discussed inmore detail in, for example, European patent application no. 0410696A1,to Kellaway et al., and U.S. Pat. No. 4,615,697, issued to Robinson,which disclosures are incorporated herein by reference.

The amount of bioadhesive agent used in the bioadhesive agent-woundhealing compositions of the present invention may vary depending uponthe type of bioadhesive employed and the particular properties desired.In general, the amount of bioadhesive agent present is the ordinarydosage required to obtain the desired result. Such dosages are known tothe skilled practitioner in the medical arts and are not a part of thepresent invention. In a preferred embodiment, the bioadhesive agent inthe bioadhesive-wound healing composition is present in an amount fromabout 0.01% to about 90%, preferably from about 0.1% to about 50%, andmore preferably from about 1% to about 2%, by weight

b. Methods for Making the Bioadhesive-Wound Healing Compositions ofEmbodiment Two (I.A-D+M11)

The present invention extends to methods for making the therapeuticbioadhesive-wound healing compositions (I.A-D+M11). In general, atherapeutic bioadhesive-wound healing composition is made by forming anadmixture of the wound healing components of Embodiment One (I.A-D) anda bioadhesive agent. In a first aspect of Embodiment Two (I.A+M11), abioadhesive-wound healing therapeutic composition is made by forming anadmixture of a bioadhesive agent and a wound healing compositioncomprising (a) a pyruvate, (b) an antioxidant, and (c) a mixture ofsaturated and unsaturated fatty acids. In a second aspect of EmbodimentTwo (I.B+M11), a bioadhesive-wound healing therapeutic composition ismade by forming an admixture of a bioadhesive agent and a wound healingcomposition comprising (a) a pyruvate, (b) a lactate, and (c) a mixtureof saturated and unsaturated fatty acids. In a third aspect ofEmbodiment Two (I.C+M11), a bioadhesive-wound healing therapeuticcomposition is made by forming an admixture of a bioadhesive agent and awound healing composition comprising (a) an antioxidant, and (b) amixture of saturated and unsaturated fatty acids. In a fourth aspect ofEmbodiment Two (I.D+M11), a bioadhesive-wound healing therapeuticcomposition is made by forming an admixture of a bioadhesive agent and awound healing composition comprising (a) a lactate, (b) an antioxidant,and (c) a mixture of saturated and unsaturated fatty acids.

In a preferred embodiment, the invention is directed to a method forpreparing a therapeutic bioadhesive-wound healing composition (I.A+M11)which comprises the steps of admixing the following ingredients:

(A) a therapeutically effective amount of a bioadhesive agent; and

(B) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

c. Methods for Employing the Bioadhesive-Wound Healing Compositions ofEmbodiment Two (I.A-D+M11)

The present invention extends to methods for employing the therapeuticbioadhesive-wound healing compositions (I.A-D+M11). In general, atherapeutic composition is employed by contacting the therapeuticcomposition with a wound. In a preferred embodiment, the invention isdirected to a method for treating a wound in a mammal with abioadhesive-wound healing composition (I.A+M11) which comprises thesteps of:

(A) providing a therapeutic bioadhesive-wound healing composition whichcomprises:

(1) a therapeutically effective amount of a bioadhesive agent; and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) contacting the bioadhesive-wound healing composition with theitching wound.

d. Augmented Bioadhesive-Wound Healing Compositions of Embodiment Two(I.A-D+M11+M)

In another aspect of Embodiment Two, the therapeutic bioadhesive-woundhealing compositions (I.A-D+M11) of the present invention may be furthercombined with medicaments useful for treating wounds (M) to formaugmented bioadhesive-wound healing compositions (I.A-D+M11+M). In thisembodiment, the combination of the bioadhesive-wound healing compositionof the present invention and the medicament useful for treating woundsprovides an augmented bioadhesive-wound healing composition having anenhanced ability to increase the proliferation and resuscitation rate ofmammalian cells. For example, the therapeutic compositions of thepresent invention may be used in combination with medicaments useful fortreating wounds such as immunostimulating agents (Betafectin™),antiviral agents, antikeratolytic agents, anti-inflammatory agents,antifungal agents, tretinoin, sunscreen agents, dermatological agents,topical antihistamine agents, antibacterial agents, other bioadhesiveagents, respiratory bursting inhibitors (lactic acid, adenosine),inhibitors of prostaglandin synthesis (ibuprofen, aspirin, indomethacin,meclofenomic acid, retinoic acid, padimate O, meclomen, oxybenzone),steroidal anti-inflammatory agents (corticosteroids including syntheticanalogs), antimicrobial agents (neosporin ointment, silvadine),antiseptic agents, anesthetic agents (pramoxine hydrochloride,lidocaine, benzocaine), cell nutrient media, burn relief medications,sun burn medications, acne preparations, insect bite and stingmedications, wound cleansers, wound dressings, scar reducing agents(vitamin E), and the like, and mixtures thereof, to further enhance theproliferation and resuscitation rate of mammalian cells. Preferably, themedicament useful for treating wounds is selected from the groupconsisting of immunostimulating agents, antiviral agents,antikeratolytic agents, anti-inflammatory agents, antifungal agents,tretinoin, sunscreen agents, dermatological agents, topicalantihistamine agents, antibacterial agents, bioadhesive agents,respiratory bursting inhibitors, inhibitors of prostaglandin synthesis,antimicrobial agents, cell nutrient media, scar reducing agents, andmixtures thereof. More preferably, the medicament useful for treatingwounds is selected from the group consisting of immunostimulatingagents, antiviral agents, antikeratolytic agents, anti-inflammatoryagents, antifungal agents, acne treating agents, sunscreen agents,dermatological agents, antihistamine agents, antibacterial agents,bioadhesive agents, and mixtures thereof.

In a preferred embodiment, the invention is directed to an augmentedbioadhesive-wound healing composition (I.A+M11+M) which comprises:

(A) a therapeutic bioadhesive-wound healing composition which comprises:

(1) a therapeutically effective amount of a bioadhesive agent; and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a medicament useful for treating wounds.

In a first aspect of this embodiment, the augmented bioadhesive-woundhealing composition is an immunostimulating bioadhesive-wound healingcomposition (I.A-D+M11+M1) which comprises an immunostimulating agent(M1), a bioadhesive agent, and a wound healing composition.Immunostimulating agents can stimulate the immune system in a patient tokill an infecting organism but do not promote the wound healing process.Applicants have found that the combination of an immunostimulatingagent, a bioadhesive agent, and a wound healing composition results in atherapeutic immunostimulating bioadhesive-wound healing compositionwhich acts synergistically to enhance wound repair in both the upper andlower portions of the skin.

In a second aspect of this embodiment, the augmented bioadhesive-woundhealing composition is an antiviral bioadhesive-wound healingcomposition (I.A-D+M11+M2) which comprises an antiviral agent (M2), abioadhesive agent, and a wound healing composition. Antiviral agents canreduce virus titers in a patient but do not promote the wound healingprocess. Applicants have found that the combination of an antiviralagent, a bioadhesive agent, and a wound healing composition results in atherapeutic antiviral bioadhesive-wound healing composition whichreduces the size, duration, and severity of oral and vaginal woundssuffered from viruses such as herpes.

In a third aspect of this embodiment, the augmented bioadhesive-woundhealing composition is an antikeratolytic bioadhesive-wound healingcomposition (I.A-D+M11+M3) which comprises an antikeratolytic agent(M3), a bioadhesive agent, and a wound healing composition.Antikeratolytic agents can reduce sealing and dryness in a patient butdo not promote the wound healing process. Applicants have found that thecombination of an antikeratolytic agent, a bioadhesive agent, and awound healing composition results in a therapeutic antikeratolyticbioadhesive-wound healing composition which reduces the duration andseverity of keratolysis type diseases such as psoriasis.

In a fourth aspect of this embodiment, the augmented bioadhesive-woundhealing composition is an anti-inflammatory bioadhesive-wound healingcomposition (I.A-D+M11+M4) which comprises anti-inflammatorybioadhesive-wound healing composition which comprises ananti-inflammatory agent (M4), a bioadhesive agent, and a wound healingcomposition. Anti-inflammatory agents can reduce inflammation in apatient but do not promote the wound healing process. Applicants havefound that the combination of an anti-inflammatory agent, a bioadhesiveagent, and a wound healing composition results in a therapeuticanti-inflammatory bioadhesive-wound healing composition which reducesthe duration and severity of inflammation.

In a fifth aspect of this embodiment, the augmented bioadhesive-woundhealing composition is an antifungal bioadhesive-wound healingcomposition (I.A-D+M11+M5) which comprises a first antifungal agent(M5), a bioadhesive agent, and a wound healing composition. The firstantifungal agent is lactic acid or sorbic acid. Antifungal agents cantreat fungal infections in a patient but do not promote the woundhealing process. Applicants have found that the combination of anantifungal agent, a bioadhesive agent, and a wound healing compositionresults in a therapeutic antifungal bioadhesive-wound healingcomposition which reduces the duration and severity of fungalinfections. The therapeutic antifungal bioadhesive-wound healingcompositions may further comprise a second antifungal agent and ananti-inflammatory agent.

In a sixth aspect of this embodiment, the augmented bioadhesive-woundhealing composition is an acne treating bioadhesive-wound healingcomposition (I.A-D+M11+M6) which comprises tretinoin (M6), a bioadhesiveagent, and a wound healing composition. Tretinoin is useful for thetopical treatment of acne vulgaris but is known to induce excessiveredness, edematous blistering or crusting, and severe local erythema andpeeling at the site of application. Applicants have found that thecombination of tretinoin, a bioadhesive agent, and a wound healingcomposition results in a therapeutic acne treating bioadhesive-woundhealing composition which reduces the duration and severity of acnevulgaris and the irritation associated with tretinoin. This inventionalso relates to methods for employing the therapeutic acne treatingbioadhesive-wound healing compositions to treat wrinkles.

In a seventh aspect of this embodiment, the augmented bioadhesive-woundhealing composition is a sunscreen treating bioadhesive-wound healingcomposition (I.A-D+M11+M7) which comprises a bioadhesive and atherapeutically effective amount of a sunscreen agent and ananti-inflammatory agent (sunscreen agent and anti-inflammatory agentcollectively referred to as M7) and the wound healing compositions ofthe present invention. Sunscreen agents can help prevent sunburn byscreening ultra violet light but do not heal injured mammalian cells.Wound healing compositions can increase the resuscitation rate ofinjured mammalian cells and the proliferation rate of new mammaliancells to replace dead cells. Wound healing compositions can alsominimize oxygen radical damage from ultra violet light. Applicants havefound that the combination of a bioadhesive agent, a sunscreen agent, ananti-inflammatory agent, and a wound healing composition results in atherapeutic sunscreen bioadhesive-wound healing compositions useful forminimizing and treating sunburn damage. The sunscreen bioadhesive-woundhealing compositions may optionally contain a therapeutically effectiveamount of a topical anesthetic to further reduce the severity ofsunburn.

In an eighth aspect of this embodiment, the augmented bioadhesive-woundhealing composition is a dermatological bioadhesive-wound healingcomposition (I.A-D+M11+M8) useful to minimize and treat diaperdermatitis. The dermatological bioadhesive-wound healing compositionscomprise a bioadhesive and a therapeutically effective amount of (1) abuffering agent to maintain the pH of the dermatitis in a range fromabout 5 to about 8 and an anti-inflammatory agent (buffering agent andanti-inflammatory agent collectively referred to as M8) and the woundhealing compositions of the present invention. Applicants have foundthat the combination of a bioadhesive, a buffering agent, ananti-inflammatory agent, and a wound healing composition results in atherapeutic dermatological bioadhesive-wound healing compositions usefulfor minimizing and treating diaper dermatitis. The dermatologicalbioadhesive-wound healing compositions may optionally contain atherapeutically effective amount of a topical antiseptic to furtherreduce the duration and severity of diaper dermatitis.

In a ninth aspect of this embodiment, the augmented bioadhesive-woundhealing composition is an antihistamine bioadhesive-wound healingcomposition (I.A-D+M11+M9) useful to minimize and treat itchingassociated with skin irritations. The antihistamine bioadhesive-woundhealing compositions comprise a therapeutically effective amount of abioadhesive, a topical antihistamine, and the wound healing compositionsof the present invention. Topical antihistamines are useful for thetopical treatment of skin irritations but are potentially sensitizing.Wound healing compositions can increase the resuscitation rate ofinjured mammalian cells and the proliferation rate of new mammaliancells to replace dead cells. Applicants have found that the combinationof a bioadhesive, a topical antihistamine, and a wound healingcomposition results in a therapeutic antihistamine bioadhesive-woundhealing composition which reduces the duration and severity of itchingassociated with skin irritations and the sensitizing effect associatedwith topical antihistamines.

In a tenth aspect of this embodiment, the augmented bioadhesive-woundhealing composition is an antibacterial bioadhesive-wound healingcomposition (I.A-D+M11+M10) which comprises a bioadhesive agent, anantibacterial agent (M10), and a wound healing composition.Antibacterial agents can treat bacterial infections in a patient but donot promote the wound healing process. Applicants have found that thecombination of a bioadhesive agent, an antibacterial agent, and a woundhealing composition results in a therapeutic antibacterialbioadhesive-wound healing composition which reduces the duration andseverity of bacterial infections.

In an eleventh aspect of this embodiment, the augmentedbioadhesive-wound healing composition is a cytoprotectivebioadhesive-wound healing composition (I.A-D+M11+X) for protectingmammalian cells from a medicament having cytotoxic properties whichcomprises a cytotoxic agent (X), a bioadhesive agent, and a woundhealing composition. Cells treated with the cytoprotective compositionsof the present invention show decreased levels of hydrogen peroxideproduction, increased resistance to cytotoxic agents, increased rates ofproliferation, and increased viability. Cytoprotective-wound healingcomposition are discussed in detail below.

The medicaments useful for treating wounds which may be employed in theaugmented bioadhesive-wound healing therapeutic compositions, such asthe immunostimulating agents, antiviral agents, antikeratolytic agents,anti-inflammatory agents, antifungal agents, tretinoin, sunscreenagents, dermatological agents, topical antihistamine agents,antibacterial agents, and the like, and the amounts which may beemployed, have been described above in detail.

The present invention extends to methods for making the augmentedbioadhesive-wound healing compositions. In general, the augmentedcompositions are made by admixing the therapeutic bioadhesive-woundhealing composition with the medicament useful for treating wounds toprepare the augmented bioadhesive-wound healing composition.

The present invention also extends to methods for employing theaugmented bioadhesive-wound healing compositions. In general, anaugmented bioadhesive-wound healing composition is employed bycontacting the composition with a wound. In a preferred embodiment, theinvention is directed to a method for treating a wound in a mammal withan augmented bioadhesive-wound healing composition (I.A+M11+M) whichcomprises the steps of:

(A) providing a therapeutic augmented bioadhesive-wound healingcomposition which comprises:

(1) a therapeutically effective amount of a bioadhesive agent;

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(3) providing a medicament useful for treating wounds; and

(B) contacting the augmented bioadhesive-wound healing composition withthe wound.

The types of wounds which may be healed using the bioadhesive-woundhealing compositions and the augmented bioadhesive-wound healingcompositions of the present invention are wounds such as those caused byviral infection, keratolysis, inflammation, fungal infections, and thelike. The therapeutic compositions may be used topically to protect andaccelerate the healing of injured tissue.

Methods for treating a wound comprise topically administering thecompositions of the present invention directly to a wound site toincrease the healing rate of the wound. The composition is maintained incontact with the wound for a period of time sufficient to increase theproliferation and resuscitation rate of the cells.

e. Formulations of the Bioadhesive-Wound Healing Compositions ofEmbodiment Two (I.A-D+M11) and (I.A-D+M11+M)

Once prepared, the inventive therapeutic bioadhesive-wound healingcompositions and augmented bioadhesive-wound healing compositions may bestored for future use or may be formulated in effective amounts withpharmaceutically acceptable carriers such as pharmaceutical appliancesand topical vehicles (oral and non-oral) to prepare a wide variety ofpharmaceutical compositions. The pharmaceutically acceptable carrierswhich may be employed and the methods used to prepare the pharmaceuticalcompositions have been described above in connection with theformulations of the wound healing compositions of Embodiment One(I.A-D).

In a preferred embodiment, the invention is directed to abioadhesive-wound healing pharmaceutical composition which comprises:

(A) a therapeutic bioadhesive-wound healing composition (I.A+M11) whichcomprises:

(1) a therapeutically effective amount of a bioadhesive agent; and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a pharmaceutically acceptable carrier selected from the groupconsisting of pharmaceutical appliances, bioadhesives, and occlusivevehicles.

In another preferred embodiment, the invention is directed to a methodfor preparing a pharmaceutical composition for increasing theproliferation and resuscitation rate of mammalian cells, which comprisesthe steps of:

(A) providing a therapeutically effective amount of a bioadhesive-woundhealing composition (I.A+M11) which comprises:

(1) a bioadhesive agent; and

(2) a wound healing composition comprising:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(B) providing a pharmaceutically acceptable carrier; and

(C) admixing the bioadhesive-wound healing composition from step (A) andthe pharmaceutically acceptable carrier from step (B) to form apharmaceutical composition.

III. Cytoprotective-Wound Healing Compositions A. Embodiment Three(I.A-D, E+X)

Applicant has discovered therapeutic cytoprotective-wound healingcompositions (I.A-D+X) which comprise a cytotoxic agent (X) and thewound healing compositions of Embodiment One (I.A-D). Preferably, thewound healing composition (I.A) comprises (a) pyruvate, (b) anantioxidant, and (c) a mixture of saturated and unsaturated fatty acids.Applicants have found that the combination of a cytotoxic agent and awound healing composition results in a cytoprotective-wound healingcomposition for protecting mammalian cells from a cytotoxic agent bypreventing and reducing injury to the cells. Cells treated with thecytoprotective-wound healing compositions of the present invention showdecreased levels of hydrogen peroxide production, increased resistanceto cytotoxic agents, increased rates of proliferation, and increasedviability. The wound healing compositions may be administered to cellsconcurrently with a cytotoxic agent or the wound healing compositionsmay be administered to cells prior to administration of a cytotoxicanticancer agent to selectively protect non-cancerous cells in thepresence of cancerous cells. Because cancerous cells have a rapidmetabolism, cancerous cells will rapidly consume the protective woundhealing composition and will not be protected by the wound healingcompositions when the chemotherapeutic medicament is subsequentlyadministered.

In another aspect of this embodiment (I.E+X), the therapeutic woundhealing composition (E) in the cytoprotective-wound healing compositionsof the present invention for treating mammalian cells, preferablyepidermal keratinocytes, comprises (a) pyruvate, and (b) an antioxidant.When the therapeutic wound healing composition in this embodiment isadministered to cells before the cytotoxic agent is administered, thecombination of pyruvate inside the cell and the antioxidant in thecellular membrane functions in a synergistic manner to reduce hydrogenperoxide production in the cell and thereby prevent injury to the cell.When injury to the cell is prevented and the cell does not requireresuscitation, the mixture of saturated and unsaturated fatty acids neednot be employed in the wound healing composition. Hence, the twocomponents in the wound healing composition in this embodiment functiontogether in a synergistic manner to prevent and reduce injury tomammalian cells.

The combination of the cytotoxic agent and the wound healingcompositions of the present invention provides a therapeutic compositionuseful for protecting mammalian cells from the cytoxic agent bypreventing and reducing injury to the cells and further increasing theresuscitation rate of injured mammalian cells. The tissue damageassociated with many cytotoxic agents is believed to be caused by theproduction of cellular produced active oxygen species. Combination ofthe cytotoxic agent and the wound healing compositions may suppress suchreactive oxygen-linked tissue injury.

The cytotoxic agents which may be used in the cytoprotective-woundhealing compositions of the present invention may be selected from awide variety of medicaments. For example, medicaments taken on a longterm regimen tend to cause liver, kidney, tissue, and other toxicityproblems. In addition, certain cytotoxic agents, such as potentchemotherapeutic medicaments used to treat malignant tissues, arebelieved to stimulate release of significant amounts of reactive oxygenspecies by mammalian tissues which can cause oxidative injury.Combination of the wound healing compositions of the present inventionwith such cytotoxic agents may inhibit induction of reactive oxygenproduction while simultaneously decreasing side effects of suchmedicaments. By decreasing the side effects of such medicaments, thedosage levels of the medicaments may be increased thereby increasing thetherapeutic effect of the medicaments. For example, the wound healingcompositions may be used in topical cytoprotective compositions incombination with cytotoxic agents such as epithelial cell cohesivenessreducers such as tretinoin (Retin A™), dermatological abradants, andanti-inflammatories, to protect and enhance the resuscitation rate ofthe injured mammalian cells. The wound healing compositions may also beused in ingestible pharmaceutical compositions in combination withmedicaments that cause cytotoxic side effects such as anti-tumor,anti-viral, and antibacterial medicaments including the lipid regulatingagents gemfibrozil and lovastatin, centrally acting anticholinesterasessuch as tacrine, chemotherapeutic medicaments such as the anthracyclineantibiotic doxorubicin, gastric irritants such as acetylsalicylic acidand ibuprofen, to protect and enhance the resuscitation rate of theinjured mammalian cells.

The cytotoxic agent in the cytoprotective-wound healing compositions maybe selected from the group consisting of doxorubicin, gemfibrozil,lovastatin, and tacrine. In a more preferred embodiment, the cytotoxicagent in the cytoprotective-wound healing compositions is selected fromthe group consisting of doxorubicin, gemfibrozil, and tacrine. In a mostpreferred embodiment, the cytotoxic agent is doxorubicin.

Doxorubicin (Adriamycin™) is a cytotoxic anthracycline antibioticreported to produce regression in disseminated neoplastic conditionssuch as in various leukemias, tumors, neuroblastomas, sarcomas, andcarcinomas. Gemfibrozil (Lopid™) is a lipid regulating agent whichlowers elevated serum lipids primarily by decreasing serum triglyceridewith a variable reduction in total serum cholesterol. Lovastatin(Mevacor™) is a cholesterol lowering agent which inhibits the enzymaticbiosynthesis of cholesterol. Tacrine (Cognex™,1,2,3,4-tetrahydro-9-acridinamine) is a centrally activeanticholinesterase useful as a cognition activator. Tacrine hasundergone clinical trials for use in treating severe Alzheimer's disease(presenile dementia).

The cytotoxic agent in the cytoprotective-wound healing compositions maybe a chemotherapeutic medicament selected from the group consisting of achemically reactive drugs having nonspecific action, anti-metabolites,antibiotics, plant products, hormones, and other miscellaneouschemotherapeutic agents. Chemically reactive drugs having nonspecificaction include alkylating agents and N-alkyl-N-nitroso compounds.Examples of alkylating agents include nitrogen mustards, azridines(ethylenimines), sulfonic acid esters, and epoxides. Anti-metabolitesare compounds that interfere with the formation or utilization of anormal cellular metabolite and include amino acid antagonists, vitaminand coenzyme antagonists, and antagonists of metabolites involved innucleic acid synthesis such as glutamine antagonists, folic acidantagonists, pyrimidine antagonists, and purine antagonists. Antibioticsare compounds produced by microorganisms that have the ability toinhibit the growth of other organisms and include actinomycins andrelated antibiotics, glutarimide antibiotics, sarkomycin, fumagillin,streptonigrin, tenuazonic acid, actinogan, peptinogan, and anthracyclicantibiotics such as doxorubicin. Plant products include colchicine,podophyllotoxin, and vinca alkaloids. Hormones include those steroidsused in breast and prostate cancer and corticosteroids used in leukemiasand lymphomas. Other miscellaneous chemotherapeutic agents includeurethan, hydroxyurea, and related compounds; thiosemicarbazones andrelated compounds; phthalanilide and related compounds; and triazenesand hydrazines. In a preferred embodiment, the anticancer agent is anantibiotic. In a more preferred embodiment, the anticancer agent isdoxorubicin. In a most preferred embodiment, the anticancer agent isdoxorubicin.

The amount of cytotoxic agent used in the present invention may varydepending upon the therapeutic dosage recommended or permitted for theparticular cytotoxic agent. In general, the amount of cytotoxic agentpresent is the ordinary dosage required to obtain the desired result.Such dosages are known to the skilled practitioner in the medical artsand are not a part of the present invention. In a preferred embodiment,the cytotoxic agent in the cytoprotective-wound healing composition ispresent in an amount from about 1% to about 50%, preferably from about10% to about 30%, and more preferably from about 20% to about 25%, byweight.

In another aspect of this embodiment, the wound healing compositions ofthe present invention may be combined in an immediate release form withan anticancer cytotoxic agent in a timed release form to provide a timedrelease cytoprotective-wound healing composition. In this embodiment,the timed release composition releases the wound healing compositionsubstantially immediately and releases the anticancer cytotoxic agentafter a suitable period of time, for example from one to 24 hours afterreleasing the wound healing composition, to selectively protectnon-cancerous cells in the presence of cancerous cells against theanticancer cytotoxic agent. Cancer cells, unlike normal cells or benigntumor cells, exhibit the properties of invasion and metastasis and arehighly anaplastic. Because cancerous cells have a rapid metabolism,cancerous cells will rapidly consume the protective therapeutic woundhealing composition and will not be protected by the therapeutic woundhealing compositions when the chemotherapeutic cytotoxic medicament issubsequently released. Non-cancerous cells which do not have such arapid metabolism will not rapidly consume the therapeutic wound healingcompositions and will be protected when the chemotherapeutic medicamentis subsequently released.

In a preferred embodiment, the invention is direct at a timed-releasecytoprotective-wound healing composition for selectively protectingnon-cancerous mammalian cells in the presence of cancerous mammaliancells from an anticancer cytotoxic agent which comprises:

(A) a therapeutically effective amount of an anticancer cytotoxic agentin timed-release form; and

(B) a wound healing composition (I.A) in immediate release formcomprising:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the resuscitation of injuredmammalian cells;

wherein the wound healing composition is released substantiallyimmediately and the anticancer cytotoxic agent is released after aperiod of time sufficient such that the cancerous cells havesubstantially metabolized the wound healing composition and thenon-cancerous cells have not substantially metabolized the wound healingcomposition.

In another preferred embodiment, the invention is direct at atimed-release cytoprotective-wound healing composition for selectivelyprotecting non-cancerous mammalian cells in the presence of cancerousmammalian cells from an anticancer cytotoxic agent which comprises:

(A) a therapeutically effective amount of an anticancer cytotoxic agentin timed-release form; and

(B) a wound healing composition (I.E) in immediate release form whichcomprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;and

(b) an antioxidant; and

wherein the wound healing composition is released substantiallyimmediately and the anticancer cytotoxic agent is released after aperiod of time sufficient such that the cancerous cells havesubstantially metabolized the wound healing composition and thenon-cancerous cells have not substantially metabolized the wound healingcomposition.

A suitable or sufficient period of time is that period of time whereinthe cancerous cells have substantially metabolized the wound healingcomposition and the non-cancerous cells have not substantiallymetabolized the wound healing composition. The period of time should notbe so long that the non-cancerous cells substantially metabolize thewound healing composition and are unprotected. The exact time is subjectto such factors as the type and quantity of wound healing compositionemployed, the cytotoxic agent used, and the type of cancerous cells andnon-cancerous cells being treated. Thus, the period of time may bevaried in order to obtain the result desired and such variations arewithin the capabilities of those skilled in the art without the need forundue experimentation.

B. Methods for Making the Cytoprotective-Wound Healing Compositions ofEmbodiment Three (I.A-D, E+X)

The present invention extends to methods for making the therapeuticcytoprotective-wound healing compositions (I.A-D, E+X). In general, atherapeutic cytoprotective-wound healing composition is made by formingan admixture of the wound healing components of Embodiment One (I.A-D)and a cytotoxic agent. In a first aspect of Embodiment Three (I.A+X), acytoprotective-wound healing therapeutic composition is made by formingan admixture of a cytotoxic agent and a wound healing compositioncomprising (a) a pyruvate, (b) an antioxidant, and (c) a mixture ofsaturated and unsaturated fatty acids. In a second aspect of EmbodimentThree (I.B +X), a cytoprotective-wound healing therapeutic compositionis made by forming an admixture of a cytotoxic agent and a wound healingcomposition comprising (a) a pyruvate, (b) a lactate, and (c) a mixtureof saturated and unsaturated fatty acids. In a third aspect ofEmbodiment Three (I.C+X), a cytoprotective-wound healing therapeuticcomposition is made by forming an admixture of a cytotoxic agent and awound healing composition comprising (a) an antioxidant, and (b) amixture of saturated and unsaturated fatty acids. In a fourth aspect ofEmbodiment Three (I.D+X), a cytoprotective-wound healing therapeuticcomposition is made by forming an admixture of a cytotoxic agent and awound healing composition comprising (a) a lactate, (b) an antioxidant,and (c) a mixture of saturated and unsaturated fatty acids. In a fifthaspect of Embodiment Three (I.E+X), a cytoprotective-wound healingtherapeutic composition is made by forming an admixture of a cytotoxicagent and a wound healing composition comprising (a) a pyruvate, and (b)an antioxidant.

In a preferred embodiment, the invention is directed to a method forpreparing a therapeutic cytoprotective-wound healing composition whichcomprises the steps of admixing the following ingredients:

(A) a therapeutically effective amount of a cytotoxic agent; and

(B) a wound healing composition (I.A) which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

In another preferred embodiment, the invention is directed to a methodfor preparing a therapeutic cytoprotective-wound healing compositionwhich comprises the steps of admixing the following ingredients:

(A) a therapeutically effective amount of a cytotoxic agent; and

(B) a wound healing composition (I.E) which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;and

(b) an antioxidant.

In a first aspect of this embodiment, a cytoprotective-wound healingcomposition is made by forming an admixture of the wound healingcomposition and the cytotoxic agent. In a second aspect of thisembodiment, a timed-release cytoprotective-wound healing composition ismade by forming an admixture of the wound healing composition inimmediate release form and the anticancer cytotoxic agent intimed-release form.

The cytoprotective-wound healing compositions may be prepared usingstandard techniques and equipment known to those skilled in the art. Theapparatus useful in accordance with the present invention comprisesapparatus well known in the chemical and biochemical arts, and thereforethe selection of the specific apparatus will be apparent to the artisan.

C. Methods for Employing the Cytoprotective-Wound Healing Compositionsof Embodiment Three (I.A-D, E+X)

The present invention extends to methods for employing the therapeuticcytoprotective-wound healing compositions. In a first aspect of thisembodiment, the wound healing compositions of the present invention maybe administered to cells concurrently with a cytotoxic agent. In asecond aspect of this embodiment, the wound healing compositions of thepresent invention may be administered to cells prior to theadministration of an anticancer cytotoxic agent to selectively protectnon-cancerous cells in the presence of cancerous cells against theanticancer agent.

In a preferred embodiment of the first aspect, the invention is directedto a method for preventing and reducing injury to mammalian cells from acytotoxic agent which comprises the steps of:

(A) providing a therapeutic cytoprotective-wound healing compositionwhich comprises:

(1) a therapeutically effective amount of a cytotoxic agent; and

(2) a wound healing composition (I.A) which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) administering the cytoprotective-wound healing composition tomammalian cells to protect the mammalian cells from the cytotoxic agent.

In another preferred embodiment of the first aspect, the invention isdirected to a method for preventing and reducing injury to mammaliancells from a cytotoxic agent which comprises the steps of:

(A) providing a therapeutic cytoprotective-wound healing compositionwhich comprises:

(1) a therapeutically effective amount of a cytotoxic agent; and

(2) a wound healing composition (I.E) which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;and

(b) an antioxidant; and

(B) administering the cytoprotective-wound healing composition tomammalian cells to protect the mammalian cells from the cytotoxic agent.

In a preferred embodiment of the second aspect, the invention is directat a method for selectively protecting non-cancerous mammalian cells inthe presence of cancerous mammalian cells from an anticancer cytotoxicagent which comprises the steps of:

(A) providing an anticancer cytotoxic agent in timed-release form; and

(B) providing a wound healing composition in an immediate release formselected from the group consisting of:

(1)

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the resuscitation of injuredmammalian cells; and

(2)

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;and

(b) an antioxidant; and

(C) administering the wound healing composition from step (B) and theanticancer cytotoxic agent from step (A) concurrently to mammalian cellsto selectively protect non-cancerous mammalian cells in the presence ofcancerous mammalian cells from the anticancer cytotoxic agent;

wherein the wound healing composition is released substantiallyimmediately and the anticancer cytotoxic agent is released after aperiod of time sufficient such that the cancerous cells havesubstantially metabolized the wound healing composition and thenon-cancerous cells have not substantially metabolized the wound healingcomposition.

In another preferred embodiment of the second aspect, the invention isdirect at a method for selectively protecting non-cancerous mammaliancells in the presence of cancerous mammalian cells from an anticancercytotoxic agent which comprises the steps of:

(A) administering to mammalian cells a wound healing composition toprevent and reduce injury to the mammalian cells selected from the groupconsisting of:

(1)

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the resuscitation of injuredmammalian cells; and

(2)

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;and

(b) an antioxidant; and

(B) waiting a period of time sufficient such that the cancerous cellshave substantially metabolized the wound healing composition and thenon-cancerous cells have not substantially metabolized the wound healingcomposition; and

(C) administering an anticancer cytotoxic agent to the mammalian cellsto treat the cancerous cells which are unprotected by the wound healingcomposition and the non-cancerous cells which are protected by the woundhealing composition to thereby increase the therapeutic effect of theanticancer cytotoxic agent.

Methods for administering the cytoprotective-wound healing compositionsof the present invention to mammalian cells will vary depending upon theparticular condition being treated and the cytotoxic agent employed. Ingeneral, the cytoprotective-wound healing compositions will beadministered in the same manner as the cytotoxic agent. Of course, thetype of carrier will vary depending upon the mode of administrationdesired for the pharmaceutical composition as is conventional in theart.

D. Augmented Cytoprotective-Wound Healing Compositions of EmbodimentThree (I.A-D, E+X+M)

In another aspect of Embodiment Three, the therapeuticcytoprotective-wound healing compositions (I.A-D, E+X) of the presentinvention may be further combined with medicaments useful for treatingwounds (M) to form augmented cytoprotective-wound healing compositions(I.A-D, E+X+M). In this embodiment, the combination of thecytoprotective-wound healing composition of the present invention andthe medicament useful for treating wounds provides an augmentedcytoprotective-wound healing composition having an enhanced ability toincrease the proliferation and resuscitation rate of mammalian cells.For example, the therapeutic compositions of the present invention maybe used in combination with medicaments useful for treating wounds suchas immunostimulating agents (Betafectin™), antiviral agents,antikeratolytic agents, anti-inflammatory agents, antifungal agents,tretinoin, sunscreen agents, dermatological agents, topicalantihistamine agents, antibacterial agents, bioadhesive agents,respiratory bursting inhibitors (lactic acid, adenosine), inhibitors ofprostaglandin synthesis (ibuprofen, aspirin, indomethacin, meclofenomicacid, retinoic acid, padimate O, meclomen, oxybenzone), steroidalanti-inflammatory agents (corticosteroids including synthetic analogs),antimicrobial agents (neosporin ointment, silvadine), antiseptic agents,anesthetic agents (pramoxine hydrochloride, lidocaine, benzocaine), cellnutrient media, burn relief medications, sun burn medications, acnepreparations, insect bite and sting medications, wound cleansers, wounddressings, scar reducing agents (vitamin E), and the like, and mixturesthereof, to further enhance the proliferation and resuscitation rate ofmammalian cells. Preferably, the medicament useful for treating woundsis selected from the group consisting of immunostimulating agents,antiviral agents, antikeratolytic agents, anti-inflammatory agents,antifungal agents, tretinoin, sunscreen agents, dermatological agents,topical antihistamine agents, antibacterial agents, bioadhesive agents,respiratory bursting inhibitors, inhibitors of prostaglandin synthesis,antimicrobial agents, cell nutrient media, scar reducing agents, andmixtures thereof. More preferably, the medicament useful for treatingwounds is selected from the group consisting of immunostimulatingagents, antiviral agents, antikeratolytic agents, anti-inflammatoryagents, antifungal agents, acne treating agents, sunscreen agents,dermatological agents, antihistamine agents, antibacterial agents,bioadhesive agents, and mixtures thereof.

In a preferred embodiment, the invention is directed to an augmentedcytoprotective-wound healing composition (I.A+X+M) which comprises:

(A) a therapeutic cytoprotective-wound healing composition whichcomprises:

(1) a therapeutically effective amount of a cytotoxic agent; and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a medicament useful for treating wounds.

The present invention extends to methods for making the augmentedcytoprotective-wound healing compositions. In general, the augmentedcompositions are made by admixing the therapeutic cytoprotective-woundhealing composition with the medicament useful for treating wounds toprepare the augmented cytoprotective-wound healing composition.

The present invention also extends to methods for employing theaugmented cytoprotective-wound healing compositions. In a first aspectof this embodiment, the wound healing compositions of the presentinvention may be administered to cells concurrently with the cytotoxicagent and the medicament useful for treating wounds. In a second aspectof this embodiment, the wound healing compositions of the presentinvention and the medicament useful for treating wounds may beadministered to cells prior to the administration of an anticancercytotoxic agent to selectively protect non-cancerous cells in thepresence of cancerous cells against the anticancer agent.

In a preferred embodiment of the first aspect, the invention is directedto a method for preventing and reducing injury to mammalian cells from acytotoxic agent which comprises the steps of:

(A) providing a therapeutic augmented cytoprotective-wound healingcomposition which comprises:

(1) a therapeutically effective amount of a cytotoxic agent; and

(2) a wound healing composition (I.A) which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(3) a medicament useful for treating wounds;

(B) administering the augmented cytoprotective-wound healing compositionto mammalian cells to protect the mammalian cells from the cytotoxicagent.

In another preferred embodiment of the first aspect, the invention isdirected to a method for preventing and reducing injury to mammaliancells from a cytotoxic agent which comprises the steps of:

(A) providing a therapeutic augmented cytoprotective-wound healingcomposition which comprises:

(1) a therapeutically effective amount of a cytotoxic agent; and

(2) a wound healing composition (I.E) which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;and

(b) an antioxidant; and

(3) a medicament useful for treating wounds;

(B) administering the augmented cytoprotective-wound healing compositionto mammalian cells to protect the mammalian cells from the cytotoxicagent.

In a preferred embodiment of the second aspect, the invention is directat a method for selectively protecting non-cancerous mammalian cells inthe presence of cancerous mammalian cells from an anticancer cytotoxicagent which comprises the steps of:

(A) providing an anticancer cytotoxic agent in timed-release form; and

(B) providing a wound healing composition in an immediate release formselected from the group consisting of:

(1)

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the resuscitation of injuredmammalian cells; and

(2)

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;and

(b) an antioxidant; and

(C) providing a medicament useful for treating wounds in an immediaterelease form; and

(D) administering the anticancer cytotoxic agent from step (A), thewound healing composition from step (B) and the medicament useful fortreating wounds from step (C) concurrently to mammalian cells toselectively protect non-cancerous mammalian cells in the presence ofcancerous mammalian cells from the anticancer cytotoxic agent;

wherein the wound healing composition and the medicament useful fortreating wounds are released substantially immediately and theanticancer cytotoxic agent is released alter a period of time sufficientsuch that the cancerous cells have substantially metabolized the woundhealing composition and the non-cancerous cells have not substantiallymetabolized the wound healing composition.

In another preferred embodiment of the second aspect, the invention isdirect at a method for selectively protecting non-cancerous mammaliancells in the presence of cancerous mammalian cells from an anticancercytotoxic agent which comprises the steps of:

(A) administering to mammalian cells a wound healing composition toprevent and reduce injury to the mammalian cells selected from the groupconsisting of:

(1)

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the resuscitation of injuredmammalian cells; and

(2)

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;and

(b) an antioxidant; and

(B) administering to mammalian cells a medicament useful for treatingwounds;

(C) waiting a period of time sufficient such that the cancerous cellshave substantially metabolized the wound healing composition and themedicament useful for treating wounds and the non-cancerous cells havenot substantially metabolized the wound healing composition and themedicament useful for treating wounds; and

(D) administering an anticancer cytotoxic agent to the mammalian cellsto treat the cancerous cells which are unprotected by the wound healingcomposition and the medicament useful for treating wounds and thenon-cancerous cells which are protected by the wound healing compositionand the medicament useful for treating wounds to thereby increase thetherapeutic effect of the anticancer cytotoxic agent.

E. Formulations of the Cytoprotective-Wound Healing Compositions ofEmbodiment Three (I.A-D, E+X) and (I.A-D, E+X+M)

Once prepared, the inventive therapeutic cytoprotective-wound healingcompositions and augmented cytoprotective-wound healing compositions maybe stored for future use or may be formulated in effective amounts withpharmaceutically acceptable carriers such as pharmaceutical appliancesand topical vehicles (oral and non-oral) to prepare a wide variety ofpharmaceutical compositions. The pharmaceutically acceptable carrierswhich may be employed and the methods used to prepare the pharmaceuticalcompositions have been described above in connection with theformulations of the wound healing compositions of Embodiment One(I.A-D). The combination of the wound healing compositions of thepresent invention and the cytotoxic agent provides acytoprotective-wound healing pharmaceutical composition having theability to prevent and reduce injury to mammalian cells from thecytotoxic agent and increase the resuscitation rate of injured mammaliancells. The dose level of the cytotoxic agent in the cytoprotective-woundhealing pharmaceutical composition may thereby be raised to higher thannormal levels.

In a preferred embodiment, the invention is directed to acytoprotective-wound healing pharmaceutical composition which comprises:

(A) a therapeutic cytoprotective-wound healing composition (I.A+X) whichcomprises:

(1) a therapeutically effective amount of a cytotoxic agent; and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a pharmaceutically acceptable carrier selected from the groupconsisting of pharmaceutical appliances, bioadhesives, and occlusivevehicles.

In another preferred embodiment, the invention is directed to a methodfor preparing a pharmaceutical composition for increasing theproliferation and resuscitation rate of mammalian cells, which comprisesthe steps of:

(A) providing a therapeutically effective amount of acytoprotective-wound healing composition (I.A+X) which comprises:

(1) a cytotoxic agent; and

(2) a wound healing composition comprising:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(B) providing a pharmaceutically acceptable carrier;, and

(C) admixing the cytoprotective-wound healing composition from step (A)and the pharmaceutically acceptable carrier from step (B) to form apharmaceutical composition.

In another embodiment, the invention is directed to a cytoprotectivecomposition for reversing the cytotoxic effects of ascorbic acid onmammalian cells which comprises ascorbic acid and a therapeuticallycytoprotective effective amount of pyruvate. The invention also extendsto a method for reversing the cytotoxic effects of ascorbic acid onmammalian cells which comprises administering to the mammalian cellsascorbic acid and a therapeutically cytoprotective effective amount ofpyruvate. A therapeutically effective amount of pyruvate is that amountof pyruvate which will reverse the cytotoxic effects of ascorbic acid onmammalian cells. In general, ascorbic acid and pyruvate are present inthe cytoprotective composition in a ratio by weight from about 1:10 toabout 10:1, preferably from about 1:5 to about 5:1, and more preferablyfrom about 1:2 to about 2:1, respectively.

F. Examples of the Cytoprotective-Wound Healing Compositions ofEmbodiment Three (I.A-D, E+X) Study 1

This study demonstrates the cytoprotective abilities of the therapeuticcytoprotective-wound healing compositions of the present invention.

Methods

Isolation of Peripheral Blood Monocytes

Peripheral blood was obtained from a normal healthy volunteer byvenipuncture using an EDTA-containing Vacutainer (Becton DickinsonMountain View, Calif.). A total of 10 ml of peripheral blood was mixedin a ratio of 1:1 with Dulbecco's Minimal Essential Medium (DMEM, GrandIsland Biologicals, GIBCO, Grand Island, N.Y.). The mixture was dividedinto 2 ml portions and each portion was layered onto 6 ml ofFicoll-Hypaque gradient mixture (Pharmacy, Inc., Piscataway, N.J.) andcentrifuged in a Beckman T-J6 refrigerated centrifuge for 30 minutes at1500 rpm and 4° C. After the cells were washed twice with phosphatebuffered saline, the cells were resuspended in Hank's Balanced SaltSolution without Ca⁺⁺ /Mg⁺⁺ (GIBCO).

Culture of U937 and Peripheral Blood Monocytes

Peripheral blood monocytes and U937 monocytic leukemia tumor cells wereplaced in sterile culture flasks and maintained in culture usingDulbecco's Minimal Essential Medium, with 10% fetal calf serum,supplemented with 2 mM glutamine and Pen/Strep. The cytotoxicity of thecytotoxic agent on the cells was analyzed by propidium iodide exclusiontechniques and flow cytometric quantitation. Viability of the cells wasquantified as the number of cells that excluded the vital dye trypanblue. Preparation of Chemicals

Sodium pyruvate was dissolved in distilled water and the solution wasadjusted to pH 7.4 with 1N sodium hydroxide solution. Solutions weresterile filtered. Stock solutions were prepared so that the vehiclewould not be more than 1% of the total volume of the culture media.

A mixture of fatty acids derived from chicken fat was prepared by mixing0.1% chicken fat with mineral oil to form an emulsified solution. Tween80 was added to separate cultures of cells at similar concentrations andto examine possible vehicle effects. α-Tocopherol phosphate (SigmaChemical Company, St. Louis, Mo.) was added directly to the culturemedium.

³ H-Thymidine Radiosotopic Incorporation Measurement of Cytotoxity

Cells were plated into 96 well dishes at a concentration of 10⁶cells/well. Tritiated thymidine (1 uCi/well) was added and the cellswere incubated for 4 hours at which time the cells were harvested usinga Cambridge cell harvestor. The samples were then placed inscintillation vials containing scintillation fluid and counted. Thesestudies yielded a measure of the ability of the cells to proliferate,which is a measure of viability.

The results from the tritiated thymidine incorporation assay, a measureof DNA synthesis and cellular proliferation, correlated directly withthe results from the dye exclusion viability assay. Because thetritiated thymidine incorporation assay is a more quantitative assay,the tritiated thymidine incorporation assay was used for the remainderof the studies.

A dose response curve for Doxorubicin (Adriamycin™) alone wasconstructed. Doxorubicin is an anthracycline antibiotic used as a firstline agent in a number of neoplastic conditions and is a wellcharacterized cytotoxic agent. Doses and times examined ranged from 0.1,0.5, 1, 5, 10, 25, and 50 ug/ml of Doxorubicin for 20-60 minutes and 24hours. The range of optimal concentrations for cytotoxity of Doxorubicinwas established for u937 monocytic minor cells to be 0.5, 1 and 5 ug at24 hours and 10 ug at 1-2 hours, see FIGS. 15A-15B and 16.

The cytoprotective agents (sodium pyruvate, Vitamin E, and fatty acids;wound healing composition) alone, and in combination, were examined fortheir ability to decrease the cytotoxity of Doxorubicin to U937monocytic leukemia cells and normal peripheral blood monocytes. Optimalconcentrations of the single ingredients of sodium pyruvate, Vitamin E,and fatty acids were examined. The optimal concentrations of the agentsthat were able to protect cells against Doxorubicin induced cytotoxitywere as follows: 10-50 U Vitamin E, 0.5% fatty acids, and 5 mM of sodiumpyruvate, see FIG. 17.

Window of susceptibility studies were conducted to determine the optimaltreatment time of the cells with the cytoprotective agents prior totreatment of the cells with the cytotoxic agent. The normal cells andU937 leukemic tumor cells were pretreated separately in "wash out"studies with the single agents alone, and in combination, at the optimalconcentration described above for various time periods, washed withfresh medium to remove the agents, and treated with the cytotoxic agent.The co-culture of normal and U937 leukemic tumor cells was treatedessentially in the same manner except that the cells were not treatedseparately, but co-cultured. The optimal pretreatment time of the cellswith the cytoprotective agents was found to be 24 hours prior totreatment of the cells with Doxorubicin. The cells were then placed inculture medium without the protective agents. The length of time thatthe cytoprotection lasted was 24 hours following Doxorubicin treatment.At this time, peripheral cell viability is a limiting factor becausethese cells are normal cells and do not remain in culture for extendedperiods of time.

Normal and U937 tumor cells were co-cultured and the cytotoxity ofDoxorubicin on the cells was determined by viability assays whichexamined the differential ability of the cytoprotective compositionsalone, and in combinations, to protect the normal cells from thecytotoxity of the chemotherapeutic agent.

The cells were isolated and examined for morphological evidence ofcytotoxicity or prevention of cytotoxicity. These studies determined thecytoprotective effect of the single agents and the combination of agentson the normal and tumor cells. DNA synthesis studies using 3H-thymidine(1 uCi/well) were carried out 4 hours prior to termination of theexperiment to determine the effect of the formulations on theproliferation of the cells as a measure of the prevention ofcytotoxicity and the extent of Doxorubicin-induced cytotoxicity.Propidium iodide exclusion analysis was carried out for directquantitation of the cytotoxicity and the prevention of cytotoxicity.Each set of studies was performed in triplicate so that statisticalanalysis of the significant differences between the treatment groupscould be conducted.

The effect of the cytoprotective agents on the co-culture of tumor andnormal cells was very different from the effect of these agents on theindividual cell types alone. An interaction between the normal cells andthe tumor cells must cause the viability of the tumor cells to besignificantly diminished. The cytoprotective combination of 5 mM sodiumpyruvate, 0.5% fatty acids, and 10 U Vitamin E provided significantprotection to the normal peripheral monocytes and did not protect thetumor cells from the effects of the cytotoxic agent.

Wash-out studies were conducted to determine viability of the peripheralblood monocytes co-cultured with U937 monocytic leukemia cells after 24hour pretreatment of the cells with the cytoprotective agents followedby administration of Doxorubicin. With no Doxorubicin treatment, theviability of the control normal peripheral cells was enhanced from 55%to 68% with the use of 5 mM sodium pyruvate and 0.5% fatty acids, seeFIG. 17. With no Doxorubicin treatment, the viability of the controlU937 cells was enhanced from 43% to 62% with the use of the combinationof the cytoprotective composition, 5 mM sodium pyruvate, 10 U Vitamin E,and 0.5% fatty acids, see FIG. 17.

Pretreatment with a combination of 10 U Vitamin E and 5 mM sodiumpyruvate prevented cytotoxity to normal peripheral blood monocytes witha concentration of 0.5 ug/ml Doxorubicin (53% to 68% viable), see FIGS.23A-23B. Pretreatment with a combination of 5 mM sodium pyruvate, 10 UVitamin E, and 0.5% fatty acids prevented cytotoxity to peripheral bloodmonocytes with a concentration of 1 ug/ml Doxorubicin (47% to 69%viable), see FIGS. 27A-27B. Pretreatment with the single agent 50 UVitamin E prevented cytotoxity to U937 tumor cells induced by 1 ug/mlDoxorubicin (42% to 62% viable), see FIGS. 21A-21B.

The viability of cultured peripheral monocytes without Doxorubicin was66% and increased to 75% with the cytoprotective combination of 5 mMsodium pyruvate, 10 U Vitamin E, and 0.5% fatty acids, see FIG. 27. Theviability of cultured peripheral monocytes treated with 0.5 ug/mlDoxorubicin was 47% and increased to 63.5% when pretreated with thecytoprotective combination of 5 mM sodium pyruvate, 10 U Vitamin E, and0.5% fatty acids, see FIG. 27. The viability of cultured peripheralmonocytes treated with 1 ug/ml Doxorubicin was 42% and increased to 66%when pretreated with the cytoprotective combination of 5 mM sodiumpyruvate, 10 U Vitamin E, and 0.5% fatty acids, see FIGS. 27A-27B.

The viability of cultured U937 tumor cells without Doxorubicin was 67%and did not increase when treated with any of the agents, see FIG. 27.The viability of cultured U937 tumor cells with 0.5 ug/ml Doxorubicintreatment was 47% and the highest increase in viability occurred withpretreatment of 50 U Vitamin E and 0.5% fatty acids, see FIG. 26. Theviability of cultured U937 tumor cells with 1 ug/ml Doxorubicintreatment was 45% and the highest increase in viability occurred withpretreatment of 10 U Vitamin E and 0.5% fatty acids, see FIGS. 26A-26B.

Optimal concentrations of the cytoprotective agents to preventDoxorubicin-induced cytotoxity were found to be 5 mM sodium pyruvate,10-50 U Vitamin E, and 0.5% fatty acids. In wash-out studies, thecytoprotective combination of sodium pyruvate, Vitamin E, and fattyacids and the combination of 5 mM sodium pyruvate and 10 U Vitamin Eprotected the normal peripheral blood monocytes from Doxorubicin-inducedcytotoxity, see FIG. 27. Vitamin E alone and fatty acids alone preventedthe cytotoxity of Doxorubicin in U937 cells, see FIGS. 19A-19B, and20A-20B. When normal peripheral blood monocytes were co-cultured withU937 monocytic leukemia tumor cells, the cytoprotective combination of 5mM sodium pyruvate, 0.5% fatty acids, and 10 U Vitamin E providedsignificant protection to the normal peripheral monocytes fromDoxorubicin-induced cytotoxity and did not protect the tumor cells fromthe effects of the cytotoxic agent, see FIGS. 38A-38B.

These results show that the combination of agents 5 mM sodium pyruvate,0.5% fatty acids, and 10 U and 50 U Vitamin E are useful as selectivecytoprotective agents for use with compounds that are toxic to normalcells as well as tumor cells.

Summary Analysis of the Data From Study 1

This is a summary analysis of the examples illustratingcytoprotective-wound healing compositions set out above to show thesynergistic effects of the wound healing components.

Peripheral blood monocytes were exposed to 0.5 μg/ml Adriamycin™ andtreated wth wound healing composition components (Sodium Pyruvate,Vitamin E, and Fatty Acids) to determine their effect on cellularviability. Adriamycin™, an anthracycline antibiotic is cytotoxic tocells. Adriamycin™ decreases cellular viability and produces cellulardeath. The wound healing composition components were tested individuallyand in combination to determine if they could reverse cellular damagecaused by Adriamycin™ and increase cellular vitality. Measurements weremade using ³ H-thymidine radioisotopic incorporation, which is a measureof DNA synthesis, i.e., cellular viability. The measure of cellularviability is the presence of living cells in the sample after treatment.

In all cases, the three component wound healing composition surpassedpredicted outcomes, clearly demonstrating unpredicted synergy. Theseresults are set out in FIG. 40.

    ______________________________________    Results               2          3               0.5 μg/ml                          0.5 μg/ml               Adriamycin ™                          Adriamycin ™    1          Treatment -                          Treatment - Percent                                        4    Treatment  Percent    Viability of  Difference    Groups     Viability of                          Cells with Wound                                        in Cellular    Viability  Controls   Healing Components                                        Viability    ______________________________________    1 - Control    54         54          0    2 - Fatty Acids                   54         47          -7        (0.5%)    3 - Vitamin E  53         50          -3        (10 units)    4 - Sodium     54         55          +1        Pyruvate        (5 mm)    5 - Pyruvate & 54         55          +1        Fatty Acids    6 - Vitamin E &                   54         64          +10        Fatty Acids    7 - Pyruvate & 55         68          +13        Vitamin E    8 - Pyruvate & 47         64          +17        Vitamin E &        Fatty Acids        (wound healing        composition)    ______________________________________     Column 1 shows the different treatment groups.     Column 2 shows the percent of living cells (viability) present when the     monocytes are treated with the cytotoxic agent, Adriamycin ™.     Column 3 shows the viability of cells treated with Adriamycin ™ and th     treatment.     Column 4 shows the change in viability from control due to the treatment.

    ______________________________________    Analysis    ______________________________________    Combination of Single Ingredient Effects    Fatty Acids (-7) & Vitamin E (-3) & Pyruvate (+1)    -9 Is The Predicted Three Component Effect    +17 Is The Wound healing composition Actual Effect    26 Is The Difference Between Predicted Effect minus Actual effect    (Synergy)    Combination of Paired and Single Ingredients    Pyruvate & Fatty Acids (+1) & vitamin E (-3)    -2 Is The Predicted Three Component Effect    +17 Is The Wound healing composition Actual Effect    19 Is The Difference between Predicted Effect minus Actual Effect    (Synergy)    Vitamin E & Fatty Acids (+10) & Pyruvate (+1)    +11 Is The Predicted Three Component Effect    +17 Is The Wound healing composition Actual Effect    6 Is The Difference between Predicted Effect minus Actual Effect    (Synergy)    Pyruvate & Vitamin E (+13) & Fatty Acids (-7)    +6 Is The Predicted Three Component Effect    +17 Is The Wound healing composition Actual Effect    11 Is The Difference between Predicted Effect minus Actual Effect    (Synergy)    ______________________________________

In all cases, the three component wound healing composition surpassedthe predicted outcomes clearly demonstrating unpredicted Synergy.

IV. Razor Cartridges Comprising Wound Healing Compositions A. EmbodimentFour (I.A-D, F+R)

Applicant has discovered therapeutic razor cartridges (I.A-D, F+R)comprising a disposable razor cartridge (R) and a wound healingcomposition delivery system affixed to the cartridge. The wound healingcomposition delivery system may comprise the wound healing compositionsof Embodiment One (I.A-D). Preferably, the wound healing composition(I.A) in the delivery system comprises (a) pyruvate, (b) an antioxidant,and (c) a mixture of saturated and unsaturated fatty acids. In anotherembodiment, the wound healing composition (I.F) comprises anantioxidant. The integral wound healing composition delivery system ispreferably in the form of a solid strip of a water-soluble encapsulatingagent comprising the wound healing composition premixed with a polymericdelivery system. Wound healing compositions can increase theresuscitation rate of injured mammalian cells and the proliferation rateof new mammalian cells to replace dead cells. Applicants have found thatbinding a wound healing composition to a razor cartridge results in atherapeutic razor cartridge which can reduce the duration and severityof shaving cuts and nicks.

The combination of the razor cartridge and the wound healingcompositions of the present invention provides a therapeutic razorcartridge useful for reducing the duration of shaving cuts andincreasing the resuscitation rate of injured mammalian cells. The tissuedamage associated with many shaving cuts and nicks is believed to becaused by the production of cellular produced active oxygen species.Combination of the therapeutic razor cartridge and the wound healingcompositions may suppress such reactive oxygen-linked tissue injury.

As set out in FIG. 50, razor cartridge 10 is typical of the type ofshaving device to which the present invention is applicable in affordinga wound healing composition delivery system which may be applieddirectly to the skin continuously with each stroke of the razor duringthe act of wet shaving. Razor cartridge 10 comprises a blade seat 12having formed thereon a guard bar 14 for smoothing the skin adjacent tothe cutting edge 16 of a razor blade 18 during shaving. Blade seat 12further includes a channel 20 which may be used to load cartridge 10upon a conventional reusable razor main frame (not shown) in thecustomary manner of sliding a receiving portion of the main frame intochannel 20 or sliding channel 20 over the receiving portion of the razormain frame. Completing the main supporting structure of razor cartridge10 and holding blade 18 in place against seat 12 is cap 22. Whilecartridge 10 has been illustrated as being of the single-blade type, itshould be understood that this structure is shown for purposes ofillustration only and that the invention to be described in detailherein is applicable not only to single-blade cartridges but equally aswell to multiple-blade shaving cartridges. The basic components ofcartridge 10 are fused, cemented, or otherwise bonded together and arecommonly referred to in the trade as bonded razor blade cartridges. Inthe embodiment of the invention illustrated in FIG. 50, a strip 24formed of an integral wound healing composition delivery system iscemented to cap 22 preferably within a recess 26 provided therefor.Strip 24 is disposed in juxtaposition with edge 16 of blade 18 andextended from a point adjacent one end of the blade to a point similarlyadjacent to the opposite end of the blade. Strip 24 may be a continuoussolid strip or a discontinuous strip comprising dots, or the like.

In addition to the wound healing composition delivery systems of thepresent invention, the razor cartridge may also contain one or more ashaving aids such as:

A. A lubricating agent for reducing the frictional forces between therazor and the skin, e.g., a microencapsulated silicone oil.

B. An agent which reduces the drag between the razor parts and theshaver's face, e.g., a polyethylene oxide in the range of molecularweights between 100,000 and 6,000,000; a non-ionic polyacrylamide;and/or a natural polysaccharide derived from plant materials such asguar gum.

C. An agent which modifies the chemical structure of the hair to allowthe razor blade to pass through the whiskers easily, e.g., a depilatoryagent.

D. A cleaning agent which allows the whisker and skin debris to bewashed more easily from the razor parts during shaving, e.g., a siliconpolyethylene oxide block copolymer and detergent such as sodium laurylsulfate.

E. A medicinal agent for killing bacteria or repairing skin damage andabrasions.

F. A cosmetic agent for softening, smoothing, conditioning, or improvingthe skin.

G. A blood coagulant for the suppression of bleeding occurring from nickand cuts.

As set out above, the configuration of the wound healing compositiondelivery system, its place of application to the razor cartridge, themanner of attachment and/or other means and method of incorporation mayvary widely to fit particular requirements and, accordingly,modifications of FIG. 50 may be made.

In accord with the present invention, the disposable razor cartridgecomprises a wound healing composition delivery system affixed to thecartridge. The wound healing composition delivery system is preferablyin the form of a solid strip of a water-soluble encapsulating agent.Encapsulating agents may be used to controllably release a large varietyof agents including various oils. Encapsulating agents are disclosed inmore detail in "Microencapsulation", pages 420-437 in "The Theory &Practice of Industrial Pharmacy", Second Edition, 1970, 1976, publishedby Lea & Febiger, a publication by Union Carbide Corporation of May1977, entitled "Polyox™, Water Soluble Resins: Forming AssociationCompounds" (the use of polyethylene oxide for microencapsulatingwater-immiscible oils at page 11 and the use of gelatin and polyethyleneoxide to form soluble films for microencapsulation applications at page17), and a publication by Union Carbide Corporation of May 1972,entitled "Polyox™, Water Soluble Resins: Thermoplastic Processing:Calendaring, Extrusion, and Injection Molding", (discloses a basicprocess for injection molding items using polyethylene oxide and theformation of calendared filing and sheets of polyethylene), whichdisclosures are incorporated herein by reference. Preferably, thewater-soluble encapsulating agent is polyethylene oxide.

U.S. Pat. Nos. 3,075,033 and 3,181,973 discloses methods for mixingpolyethylene oxide with an insoluble thermoplastic (such as polystyreneof which cap 22 and seat 12 may be made) and then forming the mixtureinto a plasticized mass. The polyethylene oxide is then released fromthe mass by the application of water.

The amount of wound healing composition used in the water-solubleencapsulating agent of the present invention may vary depending upon thetherapeutic dosage recommended or permitted. In general, the amount ofwound healing composition present is the ordinary dosage required toobtain the desired result. In a preferred embodiment, the wound healingcomposition is present in the water-soluble encapsulating agent in anamount from about 0.01% to about 30%, preferably from about 0.1% toabout 15%, and more preferably from about 1% to about 10%, by weight.

As set out above, the wound healing composition may also be firstpremixed with a polymeric delivery system. Polymeric delivery systemscan be used to control the delivery of liquids. Polymeric deliverysystems are capable of adsorbing high levels of lipophilic materials andthereafter controllably releasing the materials. Suitable polymericdelivery system include porous polymeric beads such as a cross-linkedpolymethacrylate copolymer.

In one preferred embodiment, the cross-linked polymethacrylate copolymeris POLYTRAP 6603 Polymer Powder, available from Dow Corning Corporation.POLYTRAP 6603 is a highly cross-linked polymethacrylate copolymer(acrylates copolymer) with high and selective oil adsorption capacityand hydrophobic surface properties. POLYTRAP 6603 Polymer Powder iscapable of quickly adsorbing high levels of lipophilic materials whilemaintaining free-flowing powder characteristics. The adsorption of theselipophilic materials is a physical phenomenon controlled by the surfacetension of the fluids on the polymer powder surface and filling of theinterstitial voids by capillary action. This adsorption characteristiccan be used to control the delivery of a fluid (converting liquids tosolids) or to adsorb a liquid from a surface. Lipophilic materials aredelivered by mechanical disruption of the agglomerate or vapor pressuredifferentials between the inside of the matrix and the outer environmentsurrounding the polymer. When rubbed on the skin, the lipophilicmaterials come into direct contact with the skin and meter out as thelipophilic materials is removed from the surface of the particle.

In another preferred embodiment, the cross-linked polymethacrylatecopolymer is MICRO SPONGE SKIN OIL ADSORBER 5640 POWDER, available fromDow Corning Corporation. MICROSPONGE SKIN OIL ADSORBER 5640 is a highlycross-linked polymethacrylate copolymer (acrylates copolymer) withselective oil and water adsorption capacity hydrophilic/hydrophobicsurface properties. MICROSPONGE SKIN OIL ADSORBER 5640 is capable ofadsorbing high levels of lipophilic materials.

The amount of wound healing composition used in the polymeric deliverysystem of the present invention may vary depending upon the therapeuticdosage recommended or permitted. In general, the amount of wound healingcomposition present is the ordinary dosage required to obtain thedesired result. In a preferred embodiment, the wound healing compositionis present in the polymeric delivery system in an amount from about 20%to about 80%, preferably from about 40% to about 70%, and morepreferably from about 50% to about 65%, by weight.

As set out above, the wound healing composition delivery system may bemicroencapsulated and mixed with a cement or binder and adhered to anappropriate surface of razor cartridge 10. These embodiments of theinvention have been selected to exemplify basic approaches to applyingthe wound healing composition delivery system either in rod, strip, orparticle form. The wound healing composition delivery system may beattached to an outer surface of a razor cartridge, recessed therein asin cartridge 10, formed as an integral part of one or more of the basiccartridge components (e.g., the guard bar or a spacer between blades ina twin-blade cartridge) and/or impregnated or dispersed in the materialfrom which one or more of the blade supporting cartridge components aremolded or otherwise formed. Preferably, the wound healing compositiondelivery system is an integral system permanently and substantiallyimmovably affixed to the cartridge.

In all cases, upon contact with the wet skin or by wetting of the razorcartridge, the wound healing composition delivery system becomesimmediately and repeatedly applied to the skin with each stroke of therazor. Thus, its intended function is performed continuously throughoutthe shaving act as opposed to the requirement of pre-shave orafter-shave treatment.

In another aspect of this embodiment (IV.F+R), the invention is directedto a disposable razor cartridge comprising:

(A) a blade seat;

(B) at least one razor blade;

(C) a cap; and

(D) a wound healing composition delivery system affixed to thecartridge, wherein the wound healing composition in the delivery systemcomprises an antioxidant (F).

In this embodiment, the antioxidant in the wound healing composition onthe razor cartridge may be selected from the group consisting of allforms of Vitamin A including retinol and 3,4-didehydoretinol, all formsof carotene including α-carotene, β-carotene, gamma-carotene, anddelta-carotene, all forms of Vitamin C including D-ascorbic acid andL-ascorbic acid, all forms of Vitamin E including α-tocopherol,β-tocopherol, gamma-tocopherol, delta-tocopherol, tocoquinone,tocotrienol, Vitamin E esters which readily undergo hydrolysis toVitamin E including Vitamin E acetate and Vitamin E succinate, andpharmaceutically acceptable Vitamin E salts such as Vitamin E phosphate,prodrugs of Vitamin A, carotene, Vitamin C, and Vitamin E,pharmaceutically acceptable salts of Vitamin A, carotene, Vitamin C, andVitamin E, and mixtures thereof.

B. Methods for Making the Razor Cartridges Comprising Wound HealingCompositions of Embodiment Four (I.A-D, F+R)

The present invention extends to methods for making the therapeuticrazor cartridges comprising the wound healing compositions (I.A-D, F+R).In general, a therapeutic razor cartridge is made by forming anadmixture of the wound healing components of the composition ofEmbodiment One (I.A-D, F) and affixing the composition to the cartridge.In a first aspect of Embodiment Four (IV.A+R), a therapeutic razorcartridge is made by affixing to the cartridge an admixture of a woundhealing composition comprising (a) a pyruvate, (b) an antioxidant, and(c) a mixture of saturated and unsaturated fatty acids. In a secondaspect of Embodiment Four (IV.B+R), a therapeutic razor cartridge ismade by affixing to the cartridge an admixture of a wound healingcomposition comprising (a) a pyruvate, (b) a lactate, and (c) a mixtureof saturated and unsaturated fatty acids. In a third aspect ofEmbodiment Four (IV.C+R), a therapeutic razor cartridge is made byaffixing to the cartridge an admixture of a wound healing compositioncomprising (a) an antioxidant, and (b) a mixture of saturated andunsaturated fatty acids. In a fourth aspect of Embodiment Four (IV.D+R),a therapeutic razor cartridge is made by affixing to the cartridge anadmixture of a wound healing composition comprising (a) a lactate, (b)an antioxidant, and (c) a mixture of saturated and unsaturated fattyacids. In a fifth aspect of Embodiment Four (IV.F+R), a therapeuticrazor cartridge is made by affixing to the cartridge a wound healingcomposition comprising an antioxidant.

In a preferred embodiment, the invention is directed to a method forpreparing a disposable razor cartridge which comprises providing acartridge comprising:

(A) a blade seat;

(B) at least one razor blade;

(C) a cap; and

affixing to the cartridge a wound healing composition delivery system,wherein the wound healing composition (I.A) in the delivery systemcomprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

C. Methods for Employing the Razor Cartridges Comprising Wound HealingCompositions of Embodiment Four (I.A-D, F+R)

The present invention extends to methods for employing the therapeuticrazor cartridges comprising wound healing compositions. In general, arazor cartridge is employed by contacting the cartridge with skin duringthe process of shaving.

In a preferred embodiment, the invention is directed to a method foremploying a disposable razor cartridge which comprises providing acartridge comprising:

(A) a blade seat;

(B) at least one razor blade;

(C) a cap; and

(D) a wound healing composition delivery system affixed to thecartridge, wherein the wound healing composition (I.A) in the deliverysystem comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and contacting thecartridge with skin during the process of shaving.

D. The Razor Cartridges Comprising Augmented Wound Healing Compositionsof Embodiment Four (I.A-D, F+R+M)

In another aspect of Embodiment Four, the therapeutic razor cartridgescomprising wound healing compositions (I.A-D, F+R) of the presentinvention may be combined with medicaments useful for treating wounds(M) to form razor cartridges comprising augmented wound healingcompositions (I.A-D, F+R+M). In this embodiment, the combination of therazor cartridges comprising a wound healing composition of the presentinvention and the medicament useful for treating wounds provides anaugmented razor cartridge comprising a wound healing composition havingan enhanced ability to increase the proliferation and resuscitation rateof mammalian cells. For example, the therapeutic compositions of thepresent invention may be used in combination with medicaments useful fortreating wounds such as immunostimulating agents (Betafectin™),antiviral agents, antikeratolytic agents, anti-inflammatory agents,antifungal agents, tretinoin, sunscreen agents, dermatological agents,topical antihistamine agents, antibacterial agents, bioadhesive agents,respiratory bursting inhibitors (lactic acid, adenosine), inhibitors ofprostaglandin synthesis (ibuprofen, aspirin, indomethacin, meclofenomicacid, retinoic acid, padimate O, meclomen, oxybenzone), steroidalanti-inflammatory agents (corticosteroids including synthetic analogs),antimicrobial agents (neosporin ointment, silvadine), antiseptic agents,anesthetic agents (pramoxine hydrochloride, lidocaine, benzocaine), cellnutrient media, burn relief medications, sun burn medications, acnepreparations, insect bite and sting medications, wound cleansers, wounddressings, scar reducing agents (vitamin E), and the like, and mixturesthereof, to further enhance the proliferation and resuscitation rate ofmammalian cells. Preferably, the medicament useful for treating woundsis selected from the group consisting of immunostimulating agents,antiviral agents, antikeratolytic agents, anti-inflammatory agents,antifungal agents, tretinoin, sunscreen agents, dermatological agents,topical antihistamine agents, antibacterial agents, bioadhesive agents,respiratory bursting inhibitors, inhibitors of prostaglandin synthesis,antimicrobial agents, cell nutrient media, scar reducing agents, andmixtures thereof. More preferably, the medicament useful for treatingwounds is selected from the group consisting of immunostimulatingagents, antiviral agents, antikeratolytic agents, anti-inflammatoryagents, antifungal agents, acne treating agents, sunscreen agents,dermatological agents, antihistamine agents, antibacterial agents,bioadhesive agents, and mixtures thereof.

In a preferred embodiment, the invention is directed to a razorcartridge comprising an augmented wound healing composition (I.A+R+M)which comprises a disposable razor cartridge comprising:

(A) a blade seat;

(B) at least one razor blade;

(C) a cap; and

(D) a wound healing composition delivery system affixed to thecartridge, wherein the wound healing composition (I.A) in the deliverysystem comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(d) a medicament useful for treating wounds.

The present invention extends to methods for making the razor cartridgescomprising an augmented wound healing composition. In general, the razorcartridges comprising an augmented wound healing composition are made byaffixing to the razor cartridge a wound healing composition and amedicament useful for treating wounds to prepare the augmented razorcartridges.

The present invention extends to methods for employing the therapeuticrazor cartridges comprising augmented wound healing compositions. Ingeneral, a razor cartridge is employed by contacting the augmentedcartridge with skin during the process of shaving. In a preferredembodiment, the invention is directed to a method for employing adisposable razor cartridge comprising an augmented wound healingcomposition which comprises providing a cartridge comprising:

(A) a blade seat;

(B) at least one razor blade;

(C) a cap; and

(D) a wound healing composition delivery system affixed to thecartridge, wherein the wound healing composition (I.A) in the deliverysystem comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(d) providing a medicament useful for treating wounds; and contactingthe cartridge with skin during the process of shaving.

The types of wounds which may be healed using the razor cartridgescomprising wound healing compositions and the augmented razor cartridgescomprising wound healing compositions of the present invention arewounds such as nicks and cuts. The therapeutic cartridges may be used toprotect and accelerate the healing of injured tissue.

E. Examples of the Razor Cartridges Comprising Wound HealingCompositions of Embodiment Four (I.A-D, F+R) Study 1

This study demonstrates a method for preparing an integral wound healingcomposition delivery system. Specifically, this study demonstrates amethod for incorporating a wound healing composition into awater-soluble encapsulating agent, wherein the wound healing compositionis premixed with a polymeric delivery system.

1. Prepare water containing 0.5% Triton X-100 (Octoxynol-9), a wettingagent manufactured by Rohm & Haas.

2. Dissolve 6 grams of sodium pyruvate into 18 grams of water containingTriton X-100.

3. Add solution from step 2 above slowly to 10 grams of Polytrap 6603Polymer Powder (cross-linked polymethacrylate copolymer manufactured byDow Corning). Addition of the solution is accomplished while thePolytrap 6603 is being stirred. When addition is complete, continue tomix for 10 minutes to insure good distribution.

4. Dry the Polytrap 6603 for 1 hour at 105° C. Weigh to verify thatmoisture has been removed.

5. Mix 6 grams of Vitamin E (Alpha-Tocopherol acetate) and 6 grams ofsunflower oil into a beaker.

6. Using the procedure described in step 3 (above), add the VitaminE/sunflower oil mixture to the dried Polytrap 6603 containing the sodiumpyruvate. When addition is completed, continue mixing an additional 10minutes to insure entrapment of the liquid into the Polytrap 6603.

7. Add 2% by weight of the Polytrap 6603 powder (containing 20% byweight each of sodium pyruvate, Vitamin E and sunflower oil) to thePolyox™ lubricating strip formulation.

A typical formulation could contain:

50% Coagulant Grade Polyox™

15% WSR-N-750 Grade Polyox™

29.9% medium impact polystyrene containing 10% by weight titaniumdioxide

5% Polytrap 6603 powder containing wound healing composition ingredients

0.1% Aloe Vera powder

All the ingredients are combined and mixed in a P-K Twin Shell blender.The formulation can then be extruded, compression molded, injectionmolded, or sequentially-injection molded. The various range of materialcombinations is relatively great and can be further defined as required.

Examples of the Razor Cartridges Comprising Wound Healing Compositionsof Embodiment Four (I.A-D, F+R) Study 2

This study demonstrates a method for preparing an integral wound healingcomposition delivery system. Specifically, this study demonstrates amethod for incorporating a wound healing composition into awater-soluble encapsulating agent, wherein the wound healing compositionis premixed with a polymeric delivery system.

1. Mix 6 grams of Vitamin E (Alpha-Tocopherol acetate) and 6 grams ofsunflower oil into a beaker.

2. Add Vitamin E/sunflower oil mixture to 10 grams of Polytrap 6603while the Polytrap 6603 is being stirred. When addition is complete,continue to mix for 10 minutes to insure good distribution.

3. Continue stirring the mixture in step 2 (above) and add 6 grams ofsodium pyruvate to the mixture.

4. Vitamin E and sunflower oil are now contained in the Polytrap 6603which has been coated with sodium pyruvate.

This mixture can then be added to a Polyox™ lubricating stripformulation in the same manner described above.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention and all suchmodifications are intended to be included within the scope of thefollowing claims.

I claim:
 1. A therapeutic wound healing composition which comprises:(a)pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;(b) an antioxidant; and (c) a mixture of saturated and unsaturated fattyacids wherein the fatty acids are those fatty acids required for theresuscitation of injured mammalian cells; wherein components a, b, and care present in amounts sufficient to synergistically enhance woundhealing.
 2. The composition according to claim 1, wherein the mammaliancells comprise epidermal keratinocytes.
 3. The composition according toclaim 1, wherein the pyruvate is selected from the group consisting ofpyruvic acid, lithium pyruvate, sodium pyruvate, potassium pyruvate,magnesium pyruvate, calcium pyruvate, zinc pyruvate, manganese pyruvate,methyl pyruvate, Alpha-ketoglutaric acid, pharmaceutically acceptablesalts of pyruvic acid, prodrugs of pyruvic acid, and mixtures thereof.4. The composition according to claim 3, wherein the pyruvate is sodiumpyruvate.
 5. The composition according to claim 1, wherein theantioxidant is selected from the group consisting of all forms ofVitamin A; all forms of carotene; all forms of Vitamin C; all forms ofVitamin E; Vitamin E esters which readily undergo hydrolysis to VitaminE; prodrugs of Vitamin A, carotene, Vitamin C, and Vitamin E;pharmaceutically acceptable salts of Vitamin A, carotene, Vitamin C, andVitamin E; and mixtures thereof.
 6. The composition according to claim5, wherein the antioxidant is Vitamin E acetate.
 7. The compositionaccording to claim 1, wherein the mixture of saturated and unsaturatedfatty acids is selected from the group consisting of animal andvegetable fats and waxes.
 8. The composition according to claim 7,wherein the mixture of saturated and unsaturated fatty acids is selectedfrom the group consisting of human fat, chicken fat, cow fat, sheep fat,horse fat, pig fat, and whale fat.
 9. The composition according to claim8, wherein the mixture of saturated and unsaturated fatty acidscomprises lauric acid, myristic acid, myristoleic acid, pentadecanoicacid, palmitic acid, palmitoleic acid, margaric acid, margaroleic acid,stearic, oleic acid, linoleic acid, linolenic acid, arachidic acid, andgadoleic acid.
 10. The composition according to claim 1, whereinpyruvate is present in the therapeutic wound healing composition in anamount from about 10% to about 50%, by weight of the therapeutic woundhealing composition.
 11. The Composition according to claim 1, whereinthe antioxidant is present in the therapeutic wound healing compositionin an amount from about 0.1% to about 40%, by weight of the therapeuticwound healing composition.
 12. The composition according to claim 1,wherein the mixture of saturated and unsaturated fatty acids is presentin the therapeutic wound healing composition in an amount from about 10%to about 50%, by weight of the therapeutic wound healing composition.13. A method for healing a wound in a mammal which comprisesadministering to a mammal in need thereof:a therapeutic wound healingcomposition which comprises:(a) pyruvate selected from the groupconsisting of pyruvic acid, pharmaceutically acceptable salts of pyruvicacid, and mixtures thereof; (c) a mixture of saturated and unsaturatedfatty acids wherein the fatty acids are those fatty acids required forthe resuscitation of injured mammalian cells; wherein components a, b,and c are present in synergistic amounts sufficient to synergisticallyenhance wound healing.
 14. An augmented wound healing composition havingan enhanced ability to prevent and reduce injury to mammalian cellswhich comprises:(A) a therapeutic wound healing composition whichcomprises:(a) pyruvate selected from the group consisting of pyruvicacid, pharmaceutically acceptable salts of pyruvic acid, and mixturesthereof; (b) an antioxidant; and (c) a mixture of saturated andunsaturated fatty acids wherein the fatty acids are those fatty acidsrequired for the repair of cellular membranes and resuscitation ofmammalian cells; wherein components a, b, and c are present in amountssufficient to synergistically enhance wound healing; and, (B) amedicament useful for treating wounds.
 15. The augmented wound healingcomposition according to claim 14, wherein the medicament is selectedfrom the group consisting of immunostimulating agents, antiviral agents,antikeratolytic agents, anti-inflammatory agents, antifungal agents,acne treating agents, sunscreen agents, dermatological agents,antihistamine agents, antibacterial agents, bioadhesive agents,respiratory bursting inhibitors, inhibitors of prostaglandin synthesis,antimicrobial agents, antiseptic agents, anesthetic agents, cellnutrient media, burn relief medications, sun burn medications, insectbite and sting medications, wound cleansers, wound dressings, scarreducing agents, and mixtures thereof.
 16. A method for healing a woundin a mammal with an augmented wound healing composition which comprisesadministering to a mammal in need thereof:an augmented wound healingcomposition which comprises:(1) a therapeutic wound healing compositionwhich comprises:(a) pyruvate selected from the group consisting ofpyruvic acid, pharmaceutically acceptable salts of pyruvic acid, andmixtures thereof; (b) an antioxidant; and (c) a mixture of saturated andunsaturated fatty acids wherein the fatty acids are those fatty acidsrequired for the repair of cellular membranes and resuscitation ofmammalian cells; wherein components a, b, and c are present in amountssufficient to synergistically enhance wound healing; and, (2) amedicament useful for treating wounds.